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Abstract Background Assisted Reproductive Technologies (ARTs) have been validated in human and animal to solve reproductive problems such as infertility, aging, genetic selection/amplification and diseases. The persistent gap in ART biomedical applications lies in recapitulating the early stage of ovarian folliculogenesis, thus providing protocols to drive the large reserve of immature follicles towards the gonadotropin-dependent phase. Tissue engineering is becoming a concrete solution to potentially recapitulate ovarian structure, mostly relying on the use of autologous early follicles on natural or synthetic scaffolds. Based on these premises, the present study has been designed to validate the use of the ovarian bioinspired patterned electrospun fibrous scaffolds fabricated with poly(ε-caprolactone) (PCL) for multiple preantral (PA) follicle development. Methods PA follicles isolated from lamb ovaries were cultured on PCL scaffold adopting a validated single-follicle protocol (Ctrl) or simulating a multiple-follicle condition by reproducing an artificial ovary engrafted with 5 or 10 PA (AO5PA and AO10PA). The incubations were protracted for 14 and 18 days before assessing scaffold-based microenvironment suitability to assist in vitro folliculogenesis (ivF) and oogenesis at morphological and functional level. Results The ivF outcomes demonstrated that PCL-scaffolds generate an appropriate biomimetic ovarian microenvironment supporting the transition of multiple PA follicles towards early antral (EA) stage by supporting follicle growth and steroidogenic activation. PCL-multiple bioengineering ivF (AO10PA) performed in long term generated, in addition, the greatest percentage of highly specialized gametes by enhancing meiotic competence, large chromatin remodeling and parthenogenetic developmental competence. Conclusions The study showcased the proof of concept for a next-generation ART use of PCL-patterned scaffold aimed to generate transplantable artificial ovary engrafted with autologous early-stage follicles or to advance ivF technologies holding a 3D bioinspired matrix promoting a physiological long-term multiple PA follicle protocol.

Abstract Introduction Androgenetic alopecia (AGA) is a prevalent, multifactorial form of hair loss involving complex aetiological factors, such as altered androgen regulation and energy metabolism. Existing treatments offer limited success, thus highlighting the need for advanced, personalised therapeutic strategies. This study focuses on correlating the genetic mechanisms of AGA with molecular targets involved in the response to current treatment modalities. Methods An anonymised database including 26,607 patients was subjected to analysis. The dataset included information on patients’ genotypes in 26 single nucleotide polymorphisms (SNPs), specifically, and diagnosed AGA grades, representing a broad range of ethnic backgrounds. Results In our sample, 64.6% of males and 35.4% of females were diagnosed with female pattern hair loss. This distribution aligns well with prior studies, thus validating the representativeness of our dataset. AGA grading was classified using the Hamilton–Norwood and Ludwig scales, although no association was found to the grade of the disease. SNP association analysis revealed eight SNPs, namely rs13283456 (PTGES2), rs523349 (SRD5A2), rs1800012 (COL1A1), rs4343 (ACE), rs10782665 (PTGFR), rs533116 (PTGDR2), rs12724719 (CRABP2) and rs545659 (PTGDR2), to be statistically significant with a p-value below 0.05. Conclusions The study establishes a preliminary association between eight specific SNPs and AGA. These genetic markers offer insights into the variability of therapeutic responses, thus underlining the importance of personalised treatment approaches. Our findings show the potential for more targeted research to understand these SNPs’ and further roles in AGA pathophysiology and in modulating treatment response.

Abstract Cross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage.

IntroductionExcessive calorie intake poses a significant threat to female fertility, leading to hormonal imbalances and reproductive challenges. Overconsumption of unhealthy fats exacerbates ovarian dysfunction, with an overproduction of reactive oxygen species causing oxidative stress, impairing ovarian follicle development and leading to irregular ovulation and premature ovarian failure. Interest in biological matrices with high antioxidant properties to combat diet-related oxidative stress has grown, as they contain various bioactive factors crucial for neutralizing free radicals potentially preventing female reproductive health. This systematic review evaluates the female reproductive impact of biological matrices in mitigating oxidative damages induced by over calory habits and, in particular, high fat diets.MethodsA comparative approach among mammalian models was utilized to interpret literature available data. This approach specifically investigates the antioxidant mechanisms of biological matrices on early and late ovarian folliculogenesis, under physiological and hormone-induced female reproductive cycle. Adhering to the PRISMA 2020 guidelines, only English-language publications from peer-reviewed international indexes were considered.ResultsThe analysis of 121 publications meeting the inclusion criteria facilitated the identification of crucial components of biological matrices. These components, including carbocyclic sugars, phytonutrients, organosulfur compounds, and vitamins, were evaluated for their impact on ovarian follicle resilience, oocyte quality, and reproductive lifespan. The detrimental effects of oxidative stress on female fertility, particularly exacerbated by high saturated fat diets, are well-documented. In vivo studies across mammalian preclinical models have underscored the potential of antioxidants derived from biological matrices to mitigate diet-induced conditions. These antioxidants enhance steroidogenesis and ovarian follicle development, thereby improving oocyte quality. Additionally, discussions within these publications emphasized the clinical significance of these biological matrices, translating research findings into practical applications for female health.ConclusionFurther research is essential to fully exploit the potential of these matrices in enhancing female reproduction and mitigating the effects of diets rich in fatty acids. This requires intensified in vitro studies and comprehensive collection of in vivo data before clinical trials. The promotion of ovarian resilience offers promising avenues for enhancing understanding and advancing female reproductive health world-wide.

Unsuccessful tendon healing leads to fibrosis and occasionally calcification. In these metaplastic drifts, the mouse AT preclinical injury model represents a robust experimental setting for studying tendon calcifications. Previously, calcium deposits were found in about 30% of tendons after 28 days post-injury. Although a neuromediated healing process has previously been documented, the expression patterns of NF200, NGF, NPY, GAL, and CGRP in mouse AT and their roles in metaplastic calcific repair remain to be explored. This study included a spatiotemporal analysis of these neuromarkers during the inflammatory phase (7 days p.i.) and the proliferative/early-remodelling phase (28 days p.i.). While the inflammatory phase is characterised by NF200 and CGRP upregulation, in the 28 days p.i., the non-calcified tendons (n = 16/24) showed overall NGF, NPY, GAL, and CGRP upregulation (compared to 7 days post-injury) and a return of NF200 expression to values similar to pre-injury. Presenting a different picture, in calcified tendons (n = 8), NF200 persisted at high levels, while NGF and NPY significantly increased, resulting in a higher NPY/CGRP ratio. Therefore, high levels of NF200 and imbalance between vasoconstrictive (NPY) and vasodilatory (CGRP) neuromarkers may be indicative of calcification. Tendon cells contributed to the synthesis of neuromarkers, suggesting that their neuro-autocrine/paracrine role is exerted by coordinating growth factors, cytokines, and neuropeptides. These findings offer insights into the neurobiological mechanisms of early tendon healing and identify new neuromarker profiles predictive of tendon healing outcomes.

Tendon diseases pose a significant challenge in regenerative medicine due to the limited healing capacity of this tissue. Successful tendon regeneration requires a combination of angiogenesis, immune response, and tenogenesis processes. An effective tendon engineering (TE) strategy must finely tune this systems’ interplay toward homeostasis.This study explores in vitro the paracrine influence of amniotic epithelial stem cells (AECs) engineered on a validated 3D electrospun PLGA scaffolds on HUVECs (angiogenesis), PBMCs/Jurkat (immune response), and AECs (tenogenic stem cell activation).The results revealed the role of scaffold's topology and topography in significantly modulating the paracrine profile of the cells. In detail, AECs basal release of bioactive molecules was boosted in the cells engineered on 3D scaffolds, in particular VEGF-D, b-FGF, RANTES, and PDGF-BB (p

Androgenetic alopecia (AGA) is the most common form of hair loss, significantly affecting both men and women worldwide. Characterized by progressive hair thinning and loss, AGA is primarily mediated by dihydrotestosterone (DHT). Recent research has identified numerous single-nucleotide polymorphisms (SNPs) associated with AGA, particularly in genes involved in androgen metabolism, prostaglandin pathways, and vasodilation. These genetic markers offer insights into AGA pathophysiology and potential therapeutic targets. Pharmacogenetics, the study of how genetic variations influence drug response, holds promise for personalized AGA treatment. Identifying SNPs that affect the efficacy of treatments like minoxidil and finasteride enables the development of tailored therapeutic strategies. For instance, genetic variants in the SRD5A2 gene, which affects DHT metabolism, can predict responsiveness to 5-alpha-reductase inhibitors. Beyond pharmacogenetics, RNA interference (RNAi) technologies, e.g., small interfering RNAs (siRNAs), present new therapeutic avenues. Studies have shown the efficacy of RNAi-based treatments in targeting androgen receptors, promoting hair growth in AGA models. Integrating genetic and pharmacogenetic research into clinical practice can transform AGA management, enhancing treatment efficacy and patient outcomes. In conclusion, genetic and pharmacogenetic insights are crucial for developing personalized treatments for AGA, while emerging RNAi technologies offer promising new interventions. These advancements represent significant steps toward more effective and individualized AGA therapies.

Adipose-derived stem cells (ADSCs) hold promise for tendon repair, even if their tenogenic plasticity and underlying mechanisms remain only partially understood, particularly in cells derived from the ovine animal model. This study aimed to characterize oADSCs during in vitro expansion to validate their phenotypic properties pre-transplantation. Moreover, their tenogenic potential was assessed using two in vitro-validated approaches: (1) teno-inductive conditioned media (CM) derived from a co-culture between ovine amniotic stem cells and fetal tendon explants, and (2) short- (48 h) and long-term (14 days) seeding on highly aligned PLGA (ha-PLGA) electrospun scaffold. Our findings indicate that oADSCs can be expanded without senescence and can maintain the expression of stemness (Sox2, Oct4, Nanog) and mesenchymal (CD29, CD166, CD44, CD90) markers while remaining negative for hematopoietic (CD31, CD45) and MHC-II antigens. Of note, oADSCs’ tendon differentiation potential greatly depended on the in vitro strategy. oADSCs exposed to CM significantly upregulated tendon-related genes (COL1, TNMD, THBS4) but failed to accumulate TNMD protein at 14 days of culture. Conversely, oADSCs seeded on ha-PLGA fleeces quickly upregulated the tendon-related genes (48 h) and in 14 days accumulated high levels of the TNMD protein into the cytoplasm of ADSCs, displaying a tenocyte-like morphology. This mechano-sensing cellular response involved a complete SOX9 downregulation accompanied by YAP activation, highlighting the efficacy of biophysical stimuli in promoting tenogenic differentiation. These findings underscore oADSCs’ long-term self-renewal and tendon differentiative potential, thus opening their use in a preclinical setting to develop innovative stem cell-based and tissue engineering protocols for tendon regeneration, applied to the veterinary field.

IntroductionTendon disorders present significant challenges in the realm of musculoskeletal diseases, affecting locomotor activity and causing pain. Current treatments often fall short of achieving complete functional recovery of the tendon. It is crucial to explore, in preclinical research, the pathways governing the loss of tissue homeostasis and its regeneration. In this context, this study aimed to establish a correlation between the unbiased locomotor activity pattern of CRL:CD1 (ICR) mice exposed to uni- or bilateral Achilles tendon (AT) experimental injuries and the key histomorphometric parameters that influence tissue microarchitecture recovery.MethodsThe study involved the phenotyping of spontaneous and voluntary locomotor activity patterns in male mice using digital ventilated cages (DVC®) with access to running wheels either granted or blocked. The mice underwent non-intrusive 24/7 long-term activity monitoring for the entire study period. This period included 7 days of pre-injury habituation followed by 28 days post-injury.Results and discussionThe results revealed significant variations in activity levels based on the type of tendon injury and access to running wheels. Notably, mice with bilateral lesions and unrestricted wheel access exhibited significantly higher activity after surgery. Extracellular matrix (ECM) remodeling, including COL1 deposition and organization, blood vessel remodeling, and metaplasia, as well as cytological tendon parameters, such as cell alignment and angle deviation were enhanced in surgical (bilateral lesion) and husbandry (free access to wheels) groups. Interestingly, correlation matrix analysis uncovered a strong relationship between locomotion and microarchitecture recovery (cell alignment and angle deviation) during tendon healing. Overall, this study highlights the potential of using mice activity metrics obtained from a home-cage monitoring system to predict tendon microarchitecture recovery at both cellular and ECM levels. This provides a scalable experimental setup to address the challenging topic of tendon regeneration using innovative and animal welfare-compliant strategies.

Regenerative medicine harnesses stem cells’ capacity to restore damaged tissues and organs. In vitro methods employing specific bioactive molecules, such as growth factors, bio-inductive scaffolds, 3D cultures, co-cultures, and mechanical stimuli, steer stem cells toward the desired differentiation pathways, mimicking their natural development. Chondrogenesis presents a challenge for regenerative medicine. This intricate process involves precise modulation of chondro-related transcription factors and pathways, critical for generating cartilage. Cartilage damage disrupts this process, impeding proper tissue healing due to its unique mechanical and anatomical characteristics. Consequently, the resultant tissue often forms fibrocartilage, which lacks adequate mechanical properties, posing a significant hurdle for effective regeneration. This review comprehensively explores studies showcasing the potential of amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs) in chondrogenic differentiation. These cells exhibit innate characteristics that position them as promising candidates for regenerative medicine. Their capacity to differentiate toward chondrocytes offers a pathway for developing effective regenerative protocols. Understanding and leveraging the innate properties of AMSCs and AECs hold promise in addressing the challenges associated with cartilage repair, potentially offering superior outcomes in tissue regeneration.

PurposeObesity is a multifactorial condition with a relevant genetic correlation. Recent advances in genomic research have identified several single nucleotide polymorphisms (SNPs) in genes such as FTO, MCM6, HLA, and MC4R, associated with obesity. This study aimed to evaluate the association of 102 SNPs with BMI and weight loss treatment response in a multi-ethnic population.MethodsThe study analyzed 9,372 patients for the correlation between SNPs and BMI (dataset A). The correlation between SNP and weight loss was accessed in 474 patients undergoing different treatments (dataset B). Patients in dataset B were further divided into 3 categories based on the type of intervention: dietary therapy, intragastric balloon procedures, or surgeries. SNP association analysis and multiple models of inheritance were performed.ResultsIn dataset A, ten SNPs, including rs9939609 (FTO), rs4988235 (MCM6), and rs2395182 (HLA), were significantly associated with increased BMI. Additionally, other four SNPs, rs7903146 (TCF7L2), (rs6511720), rs5400 (SLC2A2), and rs7498665 (SH2B1), showed sex-specific correlation. For dataset B, SNPs rs2016520 (PPAR-Delta) and rs2419621 (ACSL5) demonstrated significant correlation with weight loss for all treatment types. In patients who adhered to dietary therapy, SNPs rs6544713 (ABCG8) and rs762551 (CYP1A2) were strongly correlated with weight loss. Patients undergoing surgical or endoscopic procedures exhibited differential correlations with several SNPs, including rs1801725 (CASR) and rs12970134 (MC4R), and weight loss.ConclusionThis study provides valuable insights into the genetic factors influencing BMI and weight loss response to different treatments. The findings highlight the potential for personalized weight management approaches based on individual genetic profiles.

The application of biomaterials on immune regenerative strategies to deal with unsolved pathologies is getting attention in the field of tissue engineering. In this context, graphene oxide (GO) has been proposed as an immune-mimetic material largely used for developing stem cell-based regenerative therapies, since it has shown to influence stem cell behavior and modulate their immune response. Similarly, amniotic epithelial stem cells (AECs) are getting an increasing clinical interest as source of stem cells due to their great plasticity and immunomodulatory paracrine activities, even though GO bio-mimetic effects still remain unknown. To this aim, GO-functionalized glass coverslips have been used for AECs culture. The results demonstrated how GO-coating is able to induce and accelerate the Epithelial-Mesenchymal Transition (EMT), in a process mediated by the intracellular activation of TGFβ1-SMAD2/3 signaling pathway. The trans-differentiation towards mesenchymal phenotype provides AECs of migratory ability and substantially changes the pattern of cytokines secretion upon inflammatory stimulus. Indeed, GO-exposed AECs enhance their pro-inflammatory interleukins production thus inducing a more efficient activation of macrophages and, at the same time, by slightly reducing their inhibitory action on peripheral blood mononuclear cells proliferation.Therefore, the adhesion of AECs on GO-functionalized surfaces might contribute to the generation of a tailored microenvironment useful to face both the phases of the inflammation, thereby fostering the regenerative process.

Summary: Epithelial-mesenchymal transition (EMT) changes cell phenotype by affecting immune properties of amniotic epithelial cells (AECs). The present study shows how the response to lipopolysaccharide of cells collected pre- (eAECs) and post-EMT (mAECs) induces changes in their transcriptomics profile. In fact, eAECs mainly upregulate genes involved in antigen-presenting response, whereas mAECs over-express soluble inflammatory mediator transcripts. Consistently, network analysis identifies CIITA and Nrf2 as main drivers of eAECs and mAECs immune response, respectively. As a consequence, the depletion of CIITA and Nrf2 impairs the ability of eAECs and mAECs to inhibit lymphocyte proliferation or macrophage-dependent IL-6 release, thus confirming their involvement in regulating immune response. Deciphering the mechanisms controlling the immune function of AECs pre- and post-EMT represents a step forward in understanding key physiological events wherein these cells are involved (pregnancy and labor). Moreover, controlling the immunomodulatory properties of eAECs and mAECs may be essential in developing potential strategies for regenerative medicine applications.

The role of drones is becoming increasingly important within current 3D survey methodologies. Their flexibility of use and the ability to acquire images from inaccessible viewpoints make them a critical instrument in multiple fields of application at both urban and architectural scales. This success is mainly due to the progressive development of technology, including data acquisition sensors, flight systems, and data processing programs.The Cultural Heritage domain is one with the most widespread and massive applications. Besides, due to the RPAS regulations in Italy, drones less than or equal to 250 g have seen a considerable expansion in use in recent years. The improved quality of the cameras and the recent introduction of flight planning has made them proper for photogrammetric applications. Recent research reports experiments in the architectural and archaeological domains aimed at verifying the metric reliability of the acquired data compared with active instruments.In archaeological surveying, drones can cover large complex areas quickly, minimizing shadow areas concentrated in the crests of walls. The case study presented is the Canossa Castle, a medieval archaeological complex close to Reggio Emilia and extended on a steep hill with rocky spurs. The work describes integrating GNSS, 3D scanners, and ultralight RPAS photogrammetry, gathering multi-scale geometric information.The integration between the different surveying techniques allowed to plan different verification moments on the metrological reliability of the multi-resolution model.Al last, the data acquired made it possible to produce complete architectural and urban representations, improving the knowledge needed to prepare the virtual reconstruction of the entire complex area. DOI: https://doi.org/10.20365/disegnarecon.29.2022.3

There is high clinical demand for the resolution of tendinopathies, which affect mainly adult individuals and animals. Tendon damage resolution during the adult lifetime is not as effective as in earlier stages where complete restoration of tendon structure and property occurs. However, the molecular mechanisms underlying tendon regeneration remain unknown, limiting the development of targeted therapies. The research aim was to draw a comparative map of molecules that control tenogenesis and to exploit systems biology to model their signaling cascades and physiological paths. Using current literature data on molecular interactions in early tendon development, species-specific data collections were created. Then, computational analysis was used to construct Tendon NETworks in which information flow and molecular links were traced, prioritized, and enriched. Species-specific Tendon NETworks generated a data-driven computational framework based on three operative levels and a stage-dependent set of molecules and interactions (embryo–fetal or prepubertal) responsible, respectively, for signaling differentiation and morphogenesis, shaping tendon transcriptional program and downstream modeling of its fibrillogenesis toward a mature tissue. The computational network enrichment unveiled a more complex hierarchical organization of molecule interactions assigning a central role to neuro and endocrine axes which are novel and only partially explored systems for tenogenesis. Overall, this study emphasizes the value of system biology in linking the currently available disjointed molecular data, by establishing the direction and priority of signaling flows. Simultaneously, computational enrichment was critical in revealing new nodes and pathways to watch out for in promoting biomedical advances in tendon healing and developing targeted therapeutic strategies to improve current clinical interventions.

The aim of the second edition of this Special Issue was to collect both review and original research articles that investigate and elucidate the possible therapeutic role of perinatal stem cells in pathological conditions, such as cardiovascular and metabolic diseases, as well as inflammatory, autoimmune, musculoskeletal, and degenerative diseases [...]

The Amalfi Coast represents a unicum in which the complexity is due to the coexistence of natural singularities and multiple traces resulting from the adaptation made by man to the harsh orography and to the scarcity of resources. Included in the World Heritage List in 1997, the area of the UNESCO perimeter is placed at the centre of the programmatic guidelines of the Management Plan which articulates the objectives around the themes of knowledge, protection, conservation, enhancement, and communication of (and for) coastal cultural heritage. Given these premises, the contribution aims to present a critical balance between the prospects for intervention and the operations concretely defined for the conservation of the cultural landscape of the Amalfi Coast, paying attention to the fate of the proto-industrial heritage (paper mills, ironworks, mills, and hydraulic works), largely abandoned and which requires targeted strategies for the recognition of specificities and the mitigation of intrinsic vulnerabilities.

Increased production rates of plastic and limited disposal methods have fed concerns regarding environmental degradation. Whilst most of the focus is on plastic litter and marine pollution, greenhouse gas emissions of plastic over its value chains are also of interest and non-trivial at the global scale. To quantify the global warming potential of the local plastics industry, a lifecycle-based carbon footprint is presented encompassing activities such as resource extraction, polymer production and conversion, recycling, and disposal stages. The South African plastics sector is estimated to have emitted 15.8 Mt CO2 eq in 2015, with the granulate production stage bearing the highest environmental load. The consumption of fossil fuel based electricity and the burning of plastic waste also contribute notably to the overall emissions. Additionally, the recycling process in 2015 saved approximately 1.4 Mt of greenhouse gas emissions. Significance: • Research has typically focused on the environmental impacts of the end-of-life stage of plastics, namely disposal and recycling. Despite growing concern, the global warming potential of the local plastics sector across its value chain has not been investigated. • Greenhouse gas emissions arising from the South African plastic sector are non-trivial and are estimated to total 15.8 Mt CO2 eq in 2015. • Amongst the lifecycle stages, the resin production process had the highest contribution in South Africa due to the country’s coal-based monomer production process.

Epithelial-mesenchymal plasticity (EMP) refers to the ability of cells to dynamically interconvert between epithelial (E) and mesenchymal (M) phenotypes, thus generating an array of hybrid E/M intermediates with mixed E and M features. Recent findings have demonstrated how these hybrid E/M rather than fully M cells play key roles in most of physiological and pathological processes involving EMT. To this regard, the onset of hybrid E/M state coincides with the highest stemness gene expression and is involved in differentiation of either normal and cancer stem cells. Moreover, hybrid E/M cells are responsible for wound healing and create a favorable immunosuppressive environment for tissue regeneration. Nevertheless, hybrid state is responsible of metastatic process and of the increasing of survival, apoptosis and therapy resistance in cancer cells. The present review aims to describe the main features and the emerging concepts regulating EMP and the formation of E/M hybrid intermediates by describing differences and similarities between cancer and normal hybrid stem cells. In particular, the comprehension of hybrid E/M cells biology will surely advance our understanding of their features and how they could be exploited to improve tissue regeneration and repair.

Global production and consumption of plastics have increased significantly in recent years. The environmental impacts associated with this trend have received growing attention internationally with single-use plastic packaging responsible for most plastic pollution. Locally, the SA Plastics Pact, the Industry Master Plan, and the National Waste Management Strategy all aim to transform the current linear sector model into a circular system by setting targets for increased collection and recycling rates and recycled content. However, the associated impacts of implementing such circular interventions have not yet been assessed across the plastics life cycle. Industrial ecology tools, material flow analysis and life cycle assessment, are used to generate mass-based indicators as well as indicators of climate damage in the form of the global warming potential. The carbon footprint of the South African plastics value chain from cradle to grave was estimated at 17.9 Mt CO2eq emissions in 2018, with 52% of these due to the local coal-based monomer production process. The end-of-life stage lacks proper waste collection for a third of the population, but contributes only 2% to the total greenhouse gas emissions, with recycling having a minimal environmental impact. Future projections of plastics production, use, disposal, and recycling for 2025 show that increasing mechanical recycling rates to achieve stated targets would start to have a significant effect on virgin polymer demand (in the order of several billion rands of sales annually) but would also reduce waste disposal by 28% relative to baseline growth and 18% below values calculated for 2018. Significance: • Despite increased attention, the flows and resulting life cycle-based carbon footprint of the plastics sector have not been evaluated on a local scale. • The carbon footprint of the South African plastics industry is sizeable at almost 18 Mt CO2eq per annum with emissions strongly associated with the linear rather than the circular stages of the value chain. • The impacts of a key circular economy intervention, namely increased recycling rates to achieve set targets include demand reduction for virgin polymer to the tune of several billion rands.

This paper aims at presenting a dialogue-oriented approach to the construction of a graph knowledge base (KB) supporting task-oriented human-machine interactions. In particular, we focus on different pragmatic scenarios, facing the Common Ground issue and arguing that knowledge bases (in the form of graphs) are needed to make a clarification and recover pieces of information when inconsistencies occur during the communicative exchange.The main contributions of this work are: 1) a flexible dialog system architecture designed to be plugged into existing service infrastructures, 2) a graph-based knowledge representation protocol to manage both dialog domain and dialog management, 3) a detailed investigation of clarification requests forms with respect to their functions. After a brief introduction (see Section 1), we present: the theoretical underpinnings of the paper and the background work (see Section 2) our system architecture (see Sections 3 and 4) and the clarification requests (CRs) issue (see Section 5); our CRs classification, and some examples in context (see Section 6).

Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques, although they have never been tested on male infertility diseases such as varicocele (VAR). The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a rat induced VAR model. To explain cell-dependent enhancement of reproductive outcomes in rats transplanted with hAECs and hAFMSCs, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment. Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages (Mφ) recruitment and a favorable anti-inflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR.

In vitro maturation (IVM) is not a routine assisted reproductive technology (ART) for oocytes collected from early antral (EA) follicles, a large source of potentially available gametes. Despite substantial improvements in IVM in the past decade, the outcomes remain low for EA-derived oocytes due to their reduced developmental competences. To optimize IVM for ovine EA-derived oocytes, a three-dimensional (3D) scaffold-mediated follicle-enclosed oocytes (FEO) system was compared with a validated cumulus-oocyte complex (COC) protocol. Gonadotropin stimulation (eCG and/or hCG) and/or somatic cell coculture (ovarian vs. extraovarian-cell source) were supplied to both systems. The maturation rate and parthenogenetic activation were significantly improved by combining hCG stimulation with ovarian surface epithelium (OSE) cells coculture exclusively on the FEO system. Based on the data, the paracrine factors released specifically from OSE enhanced the hCG-triggering of oocyte maturation mechanisms by acting through the mural compartment (positive effect on FEO and not on COC) by stimulating the EGFR signaling. Overall, the FEO system performed on a developed reproductive scaffold proved feasible and reliable in promoting a synergic cytoplasmatic and nuclear maturation, offering a novel cultural strategy to widen the availability of mature gametes for ART.

The conservation of abandoned urban sites, increasingly widespread all over the Italian territory and mainly along the mountain slopes, represents a complex cultural challenge which includes cultural, psychological, economic, technical, as well as aesthetic and social issues. While the sudden or progressive abandonment has guaranteed, in some ways, the preservation of authentic material values, at the same time the possibility of exploiting the places again through the restoration of architecture and the improvement of conditions of accessibility is functional also for the protection of the built heritage and the intangible values that characterize it. Starting from this, the contribution focuses on a synoptic framework of interventions and strategies aimed at the redevelopment of partially or totally abandoned urban sites in Italy, concentrating attention on the goals of diverse choices for safeguarding historic heritage. The framework aims at defining an atlas of practices that, within the complex restorative approach, can provide useful cultural stimuli to calibrate interventions respecting the multiplicity of instances and values involved in historic heritage. Specific focus is, therefore, placed on the paradigmatic case of the abandoned village of Tocco Caudio near Benevento, the subject of a research program undertaken between 2012 and 2017 at the University of Naples Federico II, characterized by the construction of a “participatory” process of possible alternatives for intervention. Borghi storici, rovine, monumenti. Esperienze e prospettive di ricerca sui centri storici abbandonati in ambito italiano La conservazione di siti urbani in abbandono, sempre più diffusi sull’intero territorio italiano e principalmente lungo le pendici montuose, rappresenta una sfida culturale complessa nella quale rientrano istanze di ordine culturale, psicologico, economico, tecnico oltre che estetico e sociale. Se il repentino o progressivo abbandono delle architetture ha garantito, per certi versi, la conservazione della materia autentica e dei valori in essa contenuti, contestualmente la possibilità di fruire nuovamente dei luoghi, attraverso il restauro delle architetture e il miglioramento delle condizioni di accessibilità, diventa funzionale anche alla protezione del patrimonio costruito e dei valori immateriali che lo caratterizzano. A partire da ciò, il contributo intende mettere a fuoco un quadro sinottico di interventi e strategie tese alla riqualificazione di siti parzialmente o totalmente abbandonati in ambito italiano, concentrando l’attenzione sugli obiettivi che sostanziano le scelte per la salvaguardia del patrimonio costituito. Tale indagine mira alla definizione di un atlante di pratiche che, entro il complesso approccio restaurativo, possa fornire stimoli culturali utili a calibrare gli interventi nel rispetto della molteplicità delle istanze e dei valori coinvolti in tale patrimonio storico. Uno specifico focus è rivolto, quindi, al caso paradigmatico del borgo abbandonato di Tocco Caudio nel Beneventano, oggetto di un programma di ricerca condotto tra il 2012 e il 2017 nell’Università di Napoli Federico II e contraddistinto da un processo “partecipato” di costruzione delle possibili alternative di intervento.

Studies have indicated that air pollution, including surface-level ozone (O3), can significantly influence the risk of chronic diseases. To better understand the carcinogenic mechanisms of air pollutants and identify predictive disease biomarkers, we examined the association between traffic-related pollutants with DNA methylation alterations and bulky DNA adducts, two biomarkers of carcinogen exposure and cancer risk, in the peripheral blood of 140 volunteers—95 traffic police officers, and 45 unexposed subjects. The DNA methylation and adduct measurements were performed by bisulfite-PCR and pyrosequencing and 32P-postlabeling assay. Airborne levels of benzo(a)pyrene [B(a)P], carbon monoxide, and tropospheric O3 were determined by personal exposure biomonitoring or by fixed monitoring stations. Overall, air pollution exposure was associated with a significant reduction (1.41 units) in global DNA methylation (95% C.I. −2.65–0.04, p = 0.026). The decrement in ALU repetitive elements was greatest in the policemen working downtown (95% C.I. −3.23–−0.49, p = 0.008). The DNA adducts were found to be significantly increased (0.45 units) in the municipal officers with respect to unexposed subjects (95% C.I. 0.02–0.88, p = 0.039), mainly in those who were controlling traffic in downtown areas (95% C.I. 0.39–1.29, p < 0.001). Regression models indicated an increment of ALU methylation at higher B(a)P concentrations (95% C.I. 0.03–0.60, p = 0.032). Moreover, statistical models showed a decrement in ALU methylation and an increment of DNA damage only above the cut-off value of 30 µg/m3 O3. A significant increment of 0.73 units of IL-6 gene methylation was also found in smokers with respect to non-smokers. Our results highlighted the role of air pollution on epigenetic alterations and genotoxic effects, especially above the target value of 30 µg/m3 surface-level O3, supporting the necessity for developing public health strategies aimed to reduce traffic-related air pollution molecular alterations.

Tendon tissue engineering aims to develop effective implantable scaffolds, with ideally the native tissue’s characteristics, able to drive tissue regeneration. This research focused on fabricating tendon-like PLGA 3D biomimetic scaffolds with highly aligned fibers and verifying their influence on the biological potential of amniotic epithelial stem cells (AECs), in terms of tenodifferentiation and immunomodulation, with respect to fleeces. The produced 3D scaffolds better resemble native tendon tissue, both macroscopically, microscopically, and biomechanically. From a biological point of view, these constructs were able to instruct AECs genotypically and phenotypically. In fact, cells engineered on 3D scaffolds acquired an elongated tenocyte-like morphology; this was different from control AECs, which retained their polygonal morphology. The boosted AECs tenodifferentiation by 3D scaffolds was confirmed by the upregulation of tendon-related genes (SCX, COL1 and TNMD) and TNMD protein expression. The produced constructs also prompted AECs’ immunomodulatory potential, both at the gene and paracrine level. This enhanced immunomodulatory profile was confirmed by a greater stimulatory effect on THP-1-activated macrophages. These biological effects have been related to the mechanotransducer YAP activation evidenced by its nuclear translocation. Overall, these results support the biomimicry of PLGA 3D scaffolds, revealing that not only fiber alignment but also scaffold topology provide an in vitro favorable tenodifferentiative and immunomodulatory microenvironment for AECs that could potentially stimulate tendon regeneration.

Female reproduction focuses mainly on achieving fully grown follicles and competent oocytes to be successfully fertilized, as well as on nourishing the developing offspring once pregnancy occurs. Current evidence demonstrates that obesity and/or high-fat diet regimes can perturbate these processes, leading to female infertility and transgenerational disorders. Since the mechanisms and reproductive processes involved are not yet fully clarified, the present review is designed as a systematic and comparative survey of the available literature. The available data demonstrate the adverse influences of obesity on diverse reproductive processes, such as folliculogenesis, oogenesis, and embryo development/implant. The negative reproductive impact may be attributed to a direct action on reproductive somatic and germinal compartments and/or to an indirect influence mediated by the endocrine, metabolic, and immune axis control systems. Overall, the present review highlights the fragmentation of the current information limiting the comprehension of the reproductive impact of a high-fat diet. Based on the incidence and prevalence of obesity in the Western countries, this topic becomes a research challenge to increase self-awareness of dietary reproductive risk to propose solid and rigorous preventive dietary regimes, as well as to develop targeted pharmacological interventions.

The aim of this review is to critically analyze the evidence provided throughout the years regarding the application of motor imagery (MI) in sport performance, focusing on the PETTLEP approach. Among the different MI approaches, in fact, the PETTLEP model takes into account many different domains for increasing the performance of athletes. These domains include physical features, the environment, task-related aspects, timing, learning, emotion, and perspective.

Worldwide, infertility affects between 10 and 15% of reproductive-aged couples. Female infertility represents an increasing health issue, principally in developing countries, as the current inclinations of delaying pregnancy beyond 35 years of age significantly decrease fertility rates. Female infertility, commonly imputable to ovulation disorders, can be influenced by several factors, including congenital malformations, hormonal dysfunction, and individual lifestyle choices, such as smoking cigarettes, stress, drug use and physical activity. Moreover, diet-related elements play an important role in the regulation of ovulation. Modern types of diet that encourage a high fat intake exert a particularly negative effect on ovulation, affecting the safety of gametes and the implantation of a healthy embryo. Identifying and understanding the cellular and molecular mechanisms responsible for diet-associated infertility might help clarify the confounding multifaceted elements of infertility and uncover novel, potentially curative treatments. In this view, this systematic revision of literature will summarize the current body of knowledge of the potential effect of high-fat diet (HFD) exposure on oocyte and follicular quality and consequent female reproductive function, with particular reference to molecular mechanisms and pathways. Inflammation, oxidative stress, gene expression and epigenetics represent the main mechanisms associated with mammal folliculogenesis and oogenesis.

Current assisted reproduction technologies (ART) are insufficient to cover the slice of the population needing to restore fertility, as well as to amplify the reproductive performance of domestic animals or endangered species. The design of dedicated reproductive scaffolds has opened the possibility to better recapitulate the reproductive 3D ovarian environment, thus potentially innovating in vitro folliculogenesis (ivF) techniques. To this aim, the present research has been designed to compare ovine preantral follicles in vitro culture on poly(epsilon-caprolactone) (PCL)-based electrospun scaffolds designed with different topology (Random vs. Patterned fibers) with a previously validated system. The ivF performances were assessed after 14 days under 3D-oil, Two-Step (7 days in 3D-oil and on scaffold), or One-Step PCL protocols (14 days on PCL-scaffold) by assessing morphological and functional outcomes. The results show that Two- and One-Step PCL ivF protocols, when performed on patterned scaffolds, were both able to support follicle growth, antrum formation, and the upregulation of follicle marker genes leading to a greater oocyte meiotic competence than in the 3D-oil system. In conclusion, the One-Step approach could be proposed as a practical and valid strategy to support a synergic follicle-oocyte in vitro development, providing an innovative tool to enhance the availability of matured gametes on an individual basis for ART purposes.

Epidemiologic studies have indicated that cruciferous vegetables can influence the cancer risk; therefore, we examined with a cross-sectional approach the correlation between the frequent consumption of the total cruciferous vegetables and the formation of bulky DNA damage, a biomarker of carcinogen exposure and cancer risk, in the Gen-Air study within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. DNA damage measurements were performed in the peripheral blood of 696 of those apparently healthy without cancer controls, including 379 never-smokers and 317 former smokers from seven European countries by the 32P-postlabeling assay. In the Gen-Air controls, the median intake of cruciferous vegetables was 6.16 (IQR 1.16–13.66) g/day, ranging from 0.37 (IQR 0–6.00) g/day in Spain to 11.34 (IQR 6.02–16.07) g/day in the UK. Based on this information, participants were grouped into: (a) high consumers (>20 g/day), (b) medium consumers (3–20 g/day) and (c) low consumers (p = 0.032); this was particularly evident in former smokers (up to a 40% change, p = 0.008). The Generalized Linear Regression models indicated an overall Mean Ratio between the high and the low consumers of 0.78 (95% confidence interval, 0.64–0.97). The current study suggests that a higher intake of cruciferous vegetables is associated with a lower level of bulky DNA adducts and supports the potential for cancer prevention strategies through dietary habit changes aimed at increasing the consumption of cruciferous vegetables.

MicroRNAs (miRNAs) are key regulators of different human processes that represent a new promising class of cancer therapeutics or therapeutic targets. Indeed, in several tumor types, including non-small-cell lung carcinoma (NSCLC), the deregulated expression of specific miRNAs has been implicated in cell malignancy. As expression levels of the oncosuppressor miR-34c-3p are decreased in NSCLC compared to normal lung, we show that reintroduction of miR-34c-3p reduces NSCLC cell survival in vitro. Further, in order to deliver the miR-34c-based therapeutic selectively to tumor cells, we took advantage of a reported nucleic acid aptamer (GL21.T) that binds and inhibits the AXL transmembrane receptor and is rapidly internalized in the target cells. By applying methods successfully used in our laboratory, we conjugated miR-34c to the GL21.T aptamer as targeting moiety for the selective delivery to AXL-expressing NSCLC cells. We demonstrate that miR-34c-3p and the GL21.T/miR-34c chimera affect NSCLC cell proliferation and are able to overcome acquired RTK-inhibitor resistance by targeting AXL receptor. Thus, the GL21.T/miR-34c chimera exerts dual inhibition of AXL at functional and transcriptional levels and represents a novel therapeutic tool for the treatment of NSCLC. Keywords: miRNA, aptamer, NSCLC, lung cancer, therapeutics

The FAO Livestock Environmental Assessment and Performance (LEAP) Partnership organised a Technical Advisory Group (TAG) to develop reference guidelines on water footprinting for livestock production systems and supply chains. The mandate of the TAG was to i) provide recommendations to monitor the environmental performance of feed and livestock supply chains over time so that progress towards improvement targets can be measured, ii) be applicable for feed and water demand of small ruminants, poultry, large ruminants and pig supply chains, iii) build on, and go beyond, the existing FAO LEAP guidelines and iv) pursue alignment with relevant international standards, specifically ISO 14040 (2006)/ISO 14044 (2006), and ISO 14046 (2014). The recommended guidelines on livestock water use address both impact assessment (water scarcity footprint as defined by ISO 14046, 2014) and water productivity (water use efficiency). While most aspects of livestock water use assessment have been proposed or discussed independently elsewhere, the TAG reviewed and connected these concepts and information in relation with each other and made recommendations towards comprehensive assessment of water use in livestock production systems and supply chains. The approaches to assess the quantity of water used for livestock systems are addressed and the specific assessment methods for water productivity and water scarcity are recommended. Water productivity assessment is further advanced by its quantification and reporting with fractions of green and blue water consumed. This allows the assessment of the environmental performance related to water use of a livestock-related system by assessing potential environmental impacts of anthropogenic water consumption (only “blue water”); as well as the assessment of overall water productivity of the system (including “green” and “blue water” consumption). A consistent combination of water productivity and water scarcity footprint metrics provides a complete picture both in terms of potential productivity improvements of the water consumption as well as minimizing potential environmental impacts related to water scarcity. This process resulted for the first time in an international consensus on water use assessment, including both the life-cycle assessment community with the water scarcity footprint and the water management community with water productivity metrics.Despite the main focus on feed and livestock production systems, the outcomes of this LEAP TAG are also applicable to many other agriculture sectors.

Regenerative medicine has greatly progressed, but tendon regeneration mechanisms and robust in vitro tendon differentiation protocols remain to be elucidated. Recently, tendon explant co-culture (CO) has been proposed as an in vitro model to recapitulate the microenvironment driving tendon development and regeneration. Here, we explored standardized protocols for production and storage of bioactive tendon-derived secretomes with an evaluation of their teno-inductive effects on ovine amniotic epithelial cells (AECs). Teno-inductive soluble factors were released in culture-conditioned media (CM) only in response to active communication between tendon explants and stem cells (CMCO). Unsuccessful tenogenic differentiation in AECs was noted when exposed to CM collected from tendon explants (CMFT) only, whereas CMCO upregulated SCXB, COL I and TNMD transcripts, in AECs, alongside stimulation of the development of mature 3D tendon-like structures enriched in TNMD and COL I extracellular matrix proteins. Furthermore, although the tenogenic effect on AECs was partially inhibited by freezing CMCO, this effect could be recovered by application of an in vivo-like physiological oxygen (2% O2) environment during AECs tenogenesis. Therefore, CMCO can be considered as a waste tissue product with the potential to be used for the development of regenerative bio-inspired devices to innovate tissue engineering application to tendon differentiation and healing.

The natural healing capacity of the tendon tissue is limited due to the hypovascular and cellular nature of this tissue. So far, several conventional approaches have been tested for tendon repair to accelerate the healing process, but all these approaches have their own advantages and limitations. Regenerative medicine and tissue engineering are interdisciplinary fields that aspire to develop novel medical devices, innovative bioscaffold, and nanomedicine, by combining different cell sources, biodegradable materials, immune modulators, and nanoparticles for tendon tissue repair. Different studies supported the idea that bioscaffolds can provide an alternative for tendon augmentation with an enormous therapeutic potentiality. However, available data are lacking to allow definitive conclusion on the use of bioscaffolds for tendon regeneration and repairing. In this review, we provide an overview of the current basic understanding and material science in the field of bioscaffolds, nanomedicine, and tissue engineering for tendon repair.

Tendinopathies are at the frontier of advanced responses to health challenges and sectoral policy targets. Cell-based therapy holds great promise for tendon disorder resolution. To verify the role of stepwise trans-differentiation of amniotic epithelial stem cells (AECs) in tendon regeneration, in the present research three different AEC subsets displaying an epithelial (eAECs), mesenchymal (mAECs), and tendon-like (tdAECs) phenotype were allotransplanted in a validated experimental sheep Achilles tendon injury model. Tissue healing was analyzed adopting a comparative approach at two early healing endpoints (14 and 28 days). All three subsets of transplanted cells were able to accelerate regeneration: mAECs with a lesser extent than eAECs and tdAECs as indicated in the summary of the total histological scores (TSH), where at day 28 eAECs and tdAECs had better significant scores with respect to mAEC-treated tendons (p < 0.0001). In addition, the immunomodulatory response at day 14 showed in eAEC-transplanted tendons an upregulation of pro-regenerative M2 macrophages with respect to mAECs and tdAECs (p < 0.0001). In addition, in all allotransplanted tendons there was a favorable IL10/IL12 compared to CTR (p < 0.001). The eAECs and tdAECs displayed two different underlying regenerative mechanisms in the tendon. The eAECs positively influenced regeneration mainly through their greater ability to convey in the host tissue the shift from pro-inflammatory to pro-regenerative responses, leading to an ordered extracellular matrix (ECM) deposition and blood vessel remodeling. On the other hand, the transplantation of tdAECs acted mainly on the proliferative phase by impacting the density of ECM and by supporting a prompt recovery, inducing a low cellularity and angle alignment of the host cell compartment. These results support the idea that AECs lay the groundwork for production of different cell phenotypes that can orient tendon regeneration through a crosstalk with the host tissue. In particular, the obtained evidence suggests that eAECs are a practicable and efficient strategy for the treatment of acute tendinopathies, thus reinforcing the grounds to move their use towards clinical practice.

The intention of this special edition is to collect review and original research articles that illustrate and stimulate growing efforts to understand the implication of perinatal stem cells in pathological conditions such as cardiovascular and metabolic diseases and inflammatory, autoimmune, musculoskeletal, and degenerative diseases [...]

The challenge of osteoarthritis (OA) is to find a minimally invasive orthobiological therapy to contrast OA progression, on inflammatory and structural fronts. The aim of the present study is to compare the effects of an intra-articular injection of three orthobiological treatments, autologous culture expanded adipose-derived mesenchymal stromal cells (ADSCs), autologous stromal vascular fraction (SVF) and allogenic culture expanded amniotic epithelial stem cells (AECs), in an animal model of OA. OA was induced in 24 sheep by bilateral lateral meniscectomy and, at 3 and 6 months post-treatment, the results were analyzed with macroscopy, histology, histomorphometry, and biochemistry. All the three treatments showed better results than control (injection of NaCl), but SVF and AECs showed superiority over ADSCs, because they induced higher cartilage regeneration and lower inflammation. SVF showed better results than AECs at 3 and 6 months. To conclude, SVF seems to be more favorable than the other biological options, because it is easily obtained and rapidly used after harvesting, with good healing potential. AECs cause no discomfort and could be also considered for the treatment of OA joints.

The usefulness of nanoparticles (NPs) in the diagnostic and/or therapeutic sector is derived from their aptitude for navigating intra- and extracellular barriers successfully and to be spatiotemporally targeted. In this context, the optimization of NP delivery platforms is technologically related to the exploitation of the mechanisms involved in the NP–cell interaction. This review provides a detailed overview of the available technologies focusing on cell–NP interaction/detection by describing their applications in the fields of cancer and regenerative medicine. Specifically, a literature survey has been performed to analyze the key nanocarrier-impacting elements, such as NP typology and functionalization, the ability to tune cell interaction mechanisms under in vitro and in vivo conditions by framing, and at the same time, the imaging devices supporting NP delivery assessment, and consideration of their specificity and sensitivity. Although the large amount of literature information on the designs and applications of cell membrane-coated NPs has reached the extent at which it could be considered a mature branch of nanomedicine ready to be translated to the clinic, the technology applied to the biomimetic functionalization strategy of the design of NPs for directing cell labelling and intracellular retention appears less advanced. These approaches, if properly scaled up, will present diverse biomedical applications and make a positive impact on human health.

Amniotic membrane (AM) is considered an important medical device with many applications in regenerative medicine. The therapeutic properties of AM are due to its resistant extracellular matrix and to the large number of bioactive molecules released by its cells. An important goal that still remains to be achieved is the identification of cultural and preservation protocols able to maintain in time the membrane morphology and the biological properties of its cells. Recently, our research group demonstrated that progesterone (P4) is crucial in preventing the loss of the epithelial phenotype of amniotic epithelial cells in vitro. Followed by this premise, it has been evaluated whether P4 may also affect AM properties in a short-term culture. Results confirm that P4 preserves AM integrity and architecture with respect to untreated AM, which showed alterations in morphology. Transmission electron microscopy (TEM) analyses demonstrate that P4 also maintains unaltered cell–cell junctions, nuclear status, and intracellular organelles. On the contrary, an untreated AM experienced an extensive cell death and a strong reduction of immunomodulatory properties, measured in terms of anti-inflammatory cytokine expression and secretion. Overall, these results could open to new strategies to ameliorate the protocols for cryopreservation and tissue culture, which represent preliminary stages of AM application in regenerative medicine.

Tendon injuries are at the frontier of innovative approaches to public health concerns and sectoral policy objectives. Indeed, these injuries remain difficult to manage due to tendon’s poor healing ability ascribable to a hypo-cellularity and low vascularity, leading to the formation of a fibrotic tissue affecting its functionality. Tissue engineering represents a promising solution for the regeneration of damaged tendons with the aim to stimulate tissue regeneration or to produce functional implantable biomaterials. However, any technological advancement must take into consideration the role of the immune system in tissue regeneration and the potential of biomaterial scaffolds to control the immune signaling, creating a pro-regenerative environment. In this context, immunoengineering has emerged as a new discipline, developing innovative strategies for tendon injuries. It aims at designing scaffolds, in combination with engineered bioactive molecules and/or stem cells, able to modulate the interaction between the transplanted biomaterial-scaffold and the host tissue allowing a pro-regenerative immune response, therefore hindering fibrosis occurrence at the injury site and guiding tendon regeneration. Thus, this review is aimed at giving an overview on the role exerted from different tissue engineering actors in leading immunoregeneration by crosstalking with stem and immune cells to generate new paradigms in designing regenerative medicine approaches for tendon injuries.

Tendon disorders represent a very common pathology in today’s population, and tendinopathies that account 30% of tendon-related injuries, affect yearly millions of people which in turn cause huge socioeconomic and health repercussions worldwide. Inflammation plays a prominent role in the development of tendon pathologies, and advances in understanding the underlying mechanisms during the inflammatory state have provided additional insights into its potential role in tendon disorders. Different cell compartments, in combination with secreted immune modulators, have shown to control and modulate the inflammatory response during tendinopathies. Stromal compartment represented by tenocytes has shown to display an important role in orchestrating the inflammatory response during tendon injuries due to the interplay they exhibit with the immune-sensing and infiltrating compartments, which belong to resident and recruited immune cells. The use of stem cells or their derived secretomes within the regenerative medicine field might represent synergic new therapeutical approaches that can be used to tune the reaction of immune cells within the damaged tissues. To this end, promising opportunities are headed to the stimulation of macrophages polarization towards anti-inflammatory phenotype together with the recruitment of stem cells, that possess immunomodulatory properties, able to infiltrate within the damaged tissues and improve tendinopathies resolution. Indeed, the comprehension of the interactions between tenocytes or stem cells with the immune cells might considerably modulate the immune reaction solving hence the inflammatory response and preventing fibrotic tissue formation. The purpose of this review is to compare the roles of distinct cell compartments during tendon homeostasis and injury. Furthermore, the role of immune cells in this field, as well as their interactions with stem cells and tenocytes during tendon regeneration, will be discussed to gain insights into new ways for dealing with tendinopathies.

Amniotic epithelial stem cells (AECs) are largely studied for their pro-regenerative properties. However, it remains undetermined if low oxygen (O2) levels that AECs experience in vivo can be of value in maintaining their biological properties after isolation. To this aim, the present study has been designed to evaluate the effects of a hypoxia-mimetic agent, cobalt chloride (CoCl2), on AECs’ stemness and angiogenic activities. First, a CoCl2 dose-effect was performed to select the concentration able to induce hypoxia, through HIF-1α stabilization, without promoting any cytotoxicity effect assessed through the analysis of cell vitality, proliferation, and apoptotic-related events. Then, the identified CoCl2 dose was evaluated on the expression and angiogenic properties of AECs’ stemness markers (OCT-4, NANOG, SOX-2) by analysing VEGF expression, angiogenic chemokines’ profiles, and AEC-derived conditioned media activity through an in vitro angiogenic xeno-assay. Results demonstrated that AECs are sensitive to the cytotoxicity effects of CoCl2. The unique concentration leading to HIF-1α stabilization and nuclear translocation was 10 µM, preserving cell viability and proliferation up to 48 h. CoCl2 exposure did not modulate stemness markers in AECs while progressively decreasing VEGF expression. On the contrary, CoCl2 treatment promoted a significant short-term release of angiogenic chemokines in culture media (CM). The enrichment in bio-active factors was confirmed by the ability of CoCl2-derived CM to induce HUVEC growth and the cells’ organization in tubule-like structures. These findings demonstrate that an appropriate dose of CoCl2 can be adopted as a hypoxia-mimetic agent in AECs. The short-term, chemical-induced hypoxic condition can be targeted to enhance AECs’ pro-angiogenic properties by providing a novel approach for stem cell-free therapy protocols.

Abstract Background In recent years, autotransplantation of cryopreserved ovarian tissue became a promising approach to preserve female fertility. The slow freezing is the most effective technique which resulted in greater live birth incidence so far. Despite that, interest to vitrification of the ovarian tissue is swiftly growing, thereby undermining the necessity for further improvements in the technique. In present study, we evaluated possibilities to increase follicle survival rates adopting innovative multi-protectoral vitrification protocols, applied to the slivers of ovarian cortex or isolated early-antral follicles, frozen individually. These experimental protocols have been compared with with validated vitrification and slow freezing ones, clinically used for female fertility preservation. Results The results showed that third tested variation of experimental vitrification protocol, with four cryoprotectants in relatively low concentrations and applied to pieces of ovarian tissue at 0 °C during equilibration, increased survival rate of ovine ovarian tissue and improved results in comparison with conventional vitrification method. This variation of experimental protocol showed significant increase in percentage of follicles with good morphology (69,3%) in comparison with only commercially available vitrification protocol for ovarian tissue (62,1%). Morphology results were confirmed by TUNEL assay. Analysis of estradiol and progesterone production by cultured individual follicles after freezing/thawing revealed that steroids secretion remained significantly higher after multi-protectoral vitrification and slow freezing protocol, when follicles after standard vitrification protocol demonstrated decline in steroidogenic activity. Conclusions The multi-protectoral approach represents a workable solution to improve vitrification outcome on ovarian tissue and isolated follicles. The reduction of individual cryoprotectants concentrations, while maintaining their sufficient cumulative level in the final freezing solution, helps to increase efficiency of the procedure. Moreover, equilibration with lower temperatures helped to decrease even further the toxic effects of cryoprotectants and preserve original quality of ovarian tissue. Therefore, multi-protectoral vitrification can be suggested as an improved method for the clinical cryopreservation of ovarian tissue.

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P4) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P4 receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P4 antagonist RU486 inhibits follicular VEGF receptors’ signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles’ microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P4 in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase.

Electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds with highly aligned fibers (ha-PLGA) represent promising materials in the field of tendon tissue engineering (TE) due to their characteristics in mimicking fibrous extracellular matrix (ECM) of tendon native tissue. Among these properties, scaffold biodegradability must be controlled allowing its replacement by a neo-formed native tendon tissue in a controlled manner. In this study, ha-PLGA were subjected to hydrolytic degradation up to 20 weeks, under di-H2O and PBS conditions according to ISO 10993-13:2010. These were then characterized for their physical, morphological, and mechanical features. In vitro cytotoxicity tests were conducted on ovine amniotic epithelial stem cells (oAECs), up to 7 days, to assess the effect of non-buffered and buffered PLGA by-products at different concentrations on cell viability and their stimuli on oAECs’ immunomodulatory properties. The ha-PLGA scaffolds degraded slowly as evidenced by a slight decrease in mass loss (14%) and average molecular weight (35%), with estimated degradation half-time of about 40 weeks under di-H2O. The ultrastructure morphology of the scaffolds showed no significant fiber degradation even after 20 weeks, but alteration of fiber alignment was already evident at week 1. Moreover, mechanical properties decreased throughout the degradation times under wet as well as dry PBS conditions. The influence of acid degradation media on oAECs was dose-dependent, with a considerable effect at 7 days’ culture point. This effect was notably reduced by using buffered media. To a certain level, cells were able to compensate the generated inflammation-like microenvironment by upregulating IL-10 gene expression and favoring an anti-inflammatory rather than pro-inflammatory response. These in vitro results are essential to better understand the degradation behavior of ha-PLGA in vivo and the effect of their degradation by-products on affecting cell performance. Indeed, buffering the degradation milieu could represent a promising strategy to balance scaffold degradation. These findings give good hope with reference to the in vivo condition characterized by physiological buffering systems.

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis (ivF) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up ivF cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D ivF on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to ivF as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.

Recently, the research on stemness and multilineage differentiation mechanisms has greatly increased its value due to the potential therapeutic impact of stem cell-based approaches. Stem cells modulate their self-renewing and differentiation capacities in response to endogenous and/or extrinsic factors that can control stem cell fate. One key factor controlling stem cell phenotype is oxygen (O2). Several pieces of evidence demonstrated that the complexity of reproducing O2 physiological tensions and gradients in culture is responsible for defective stem cell behavior in vitro and after transplantation. This evidence is still worsened by considering that stem cells are conventionally incubated under non-physiological air O2 tension (21%). Therefore, the study of mechanisms and signaling activated at lower O2 tension, such as those existing under native microenvironments (referred to as hypoxia), represent an effective strategy to define if O2 is essential in preserving naïve stemness potential as well as in modulating their differentiation. Starting from this premise, the goal of the present review is to report the status of the art about the link existing between hypoxia and stemness providing insight into the factors/molecules involved, to design targeted strategies that, recapitulating naïve O2 signals, enable towards the therapeutic use of stem cell for tissue engineering and regenerative medicine.

Abstract The in vitro expansion is detrimental to therapeutic applications of amniotic epithelial cells (AEC), an emerging source of fetal stem cells. This study provides molecular evidences of progesterone (P4) role in preventing epithelial-mesenchymal transition (EMT) in ovine AEC (oAEC). oAEC amplified under standard conditions spontaneously acquired mesenchymal properties through the up-regulation of EMT-transcription factors. P4 supplementation prevented phenotype shift by inhibiting the EMT-inducing mechanism such as the autocrine production of TGF-β and the activation of intracellular-related signaling. The effect of P4 still persisted for one passage after steroid removal from culture as well as steroid supplementation promptly reversed mesenchymal phenotype in oAEC which have experienced EMT during amplification. Furthermore, P4 promoted an acute up-regulation of pluripotent genes whereas enhanced basal and LPS-induced oAEC anti-inflammatory response with an increase in anti-inflammatory and a decrease in pro-inflammatory cytokines expression. Altogether, these results indicate that P4 supplementation is crucial to preserve epithelial phenotype and to enhance biological properties in expanded oAEC. Therefore, an innovative cultural approach is proposed in order to improve therapeutic potential of this promising source of epithelial stem cells.

Amniotic epithelial cells (AECs), an emerging source of extrafoetal stem cells, have recently attracted attention for their great regenerative potential. Since AEC amplifications are accompanied by the loss of their native epithelial phenotype and by the progressive reduction of relevant biological properties, the issue to be addressed is the development of effective culture protocols. In this context, recently, it has been demonstrated that progesterone (P4) supplementation during ovine AEC (oAEC) expansion could prevent the undesirable epithelial-mesenchymal transition (EMT). In contrast, there is no information to date on the role of the other pregnancy steroids in culture. With this aim, the present study has been designed to clarify the impact of estradiol (E2), alone or in combination with P4 (12.5 μM and 25 μM), during oAEC amplification. Steroid supplementations were assessed by testing oAEC proliferation, stemness, EMT, and osteogenic or chondrogenic plasticity. The results indicated that EMT can be prevented exclusively in the presence of high doses of P4, while it occurred rapidly in cells exposed to E2 as denoted by protein (cytokeratin-8 and alpha-SMA) and gene expression (vimentin and snail) profiles. Moreover, steroid exposure was able to influence highly oAEC plasticity. Particularly, P4-treated cells displayed a precommitment towards osteogenic lineage, confirmed by the upregulation of OCN, RUNX2, and the greater deposition of calcium nodules. Conversely, P4 exposure inhibited oAEC chondrogenic differentiation, which was induced in E2-treated cells as confirmed by the upregulation of chondrogenesis-related genes (SOX9, ACAN, and COL2A1) and by the accumulation of Alcian blue-positive extracellular matrix. Simultaneously, E2-treated cells remained unresponsive to osteogenic inductive stimuli. In conclusion, media supplementation with high doses of steroids may be adopted to modulate phenotype and plasticity during oAEC amplification. Relevantly, the osteo or chondro steroid-induced precommitment may open unprecedented cell-based therapies to face the unsolved orthopaedic issues related to osteochondral regeneration.