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Initially observed on synthetic nanoparticles, the existence of biomolecular corona and its role in determining nanoparticle identity and function are now beginning to be acknowledged in biogenic nanoparticles, particularly in extracellular vesicles - membrane-enclosed nanoparticle shuttling proteins, nucleic acids, and metabolites which are released by cells for physiological and pathological communication - we developed a methodology based on fluorescence correlation spectroscopy to track biomolecular corona formation on extracellular vesicles derived from human red blood cells and amniotic membrane mesenchymal stromal cells when these vesicles are dispersed in human plasma. The methodology allows for tracking corona dynamics in situ under physiological conditions. Results evidence that the two extracellular vesicle populations feature distinct corona dynamics. These findings indicate that the dynamics of the biomolecular corona may ultimately be linked to the cellular origin of the extracellular vesicles, revealing an additional level of heterogeneity, and possibly of bionanoscale identity, that characterizes circulating extracellular vesicles.

Graphene oxide (GO), a carbon-based nanomaterial, presents significant potential across biomedical fields such as bioimaging, drug delivery, biosensors, and phototherapy. This study examines the effects of integrating GO into poly(lactic-co-glycolic acid) (PLGA) scaffolds on human immune cell function. Our results demonstrate that high concentrations of GO reduce the viability of peripheral blood mononuclear cells (PBMCs) following stimulation with anti-CD3 antibody. This reduction extends to T lymphocyte activation, evident from the diminished proliferative response to T cell receptor engagement and impaired differentiation into T helper subsets and regulatory T cells. Interestingly, although GO induces a minimal response in resting monocytes, but it significantly affects both the viability and the differentiation potential of monocytes induced to mature toward M1 pro-inflammatory and M2-like immunoregulatory macrophages. This study seeks to address a critical gap by investigating the in vitro immunomodulatory effects of PLGA scaffolds incorporating various concentrations of GO on primary immune cells, specifically PBMCs isolated from healthy donors. Our findings emphasize the need to optimize the GO to PLGA ratios and scaffold design to advance PLGA-GO-based biomedical applications. STATEMENT OF SIGNIFICANCE: Graphene oxide (GO) holds immense promise for biomedical applications due to its unique properties. However, concerns regarding its potential to trigger adverse immune responses remain. This study addresses this critical gap by investigating the in vitro immunomodulatory effects of PLGA scaffolds incorporating increasing GO concentrations on human peripheral blood mononuclear cells (PBMCs). By elucidating the impact on cell viability, T cell proliferation and differentiation, and the maturation/polarization of antigen-presenting cells, this work offers valuable insights for designing safe and immunologically compatible GO-based biomaterials for future clinical translation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells ( h TSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-β1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased h TSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased h TSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension. This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities. This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes., Competing Interests: Funding The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: Ornella Parolini reports financial support was provided by PRIN 2017 program of Italian Ministry of Research and University (MIUR, Grant No. 2017RSAFK7). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The intricate interplay between the developing placenta and fetal-maternal interactions is critical for pregnancy outcomes. Despite advancements, gaps persist in understanding biomechanics, transport processes, and blood circulation parameters, all of which are crucial for safe pregnancies. Moreover, the complexity of fetal-maternal interactions led to conflicting data and methodological variations. This review presents a comprehensive overview of current knowledge on fetal-maternal interface structures, with a particular focus on the first trimester. More in detail, the embryological development, structural characteristics, and physiological functions of placental chorionic plate and villi, fetal membranes and umbilical cord are discussed. Furthermore, a description of the main structures and features of maternal and fetal fluid dynamic exchanges is provided. However, ethical constraints and technological limitations pose still challenges to studying early placental development directly, which calls for sophisticated in vitro , microfluidic organotypic models for advancing our understanding. For this, knowledge about key in vivo parameters are necessary for their design. In this scenario, the integration of data from later gestational stages and mathematical/computational simulations have proven to be useful tools. Notwithstanding, further research into cellular and molecular mechanisms at the fetal-maternal interface is essential for enhancing prenatal care and improving maternal and fetal health outcomes., Competing Interests: Author MG was employed by Seqvera Ltd. Author RG-V was employed by Evercyte GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Masserdotti, Gasik, Grillari-Voglauer, Grillari, Cargnoni, Chiodelli, Papait, Magatti, Romoli, Ficai, Di Pietro, Lattanzi, Silini and Parolini.)

Surgically addressing tumors poses a challenge, requiring a tailored, multidisciplinary approach for each patient based on the unique aspects of their case. Innovative therapeutic regimens combined to reliable reconstructive methods can contribute to an extended patient's life expectancy. This study presents a detailed comparative investigation of near-infrared therapy protocols, examining the impact of non-fractionated and fractionated irradiation regimens on cancer treatment. The therapy is based on the implantation of graphene oxide/poly(lactic-co-glycolic acid) three-dimensional printed scaffolds, exploring their versatile applications in oncology by the examination of pro-inflammatory cytokine secretion, immune response, and in vitro and in vivo tumor therapy. The investigation into cell death patterns (apoptosis vs necrosis) underlines the pivotal role of protocol selection underscores the critical influence of treatment duration on cell fate, establishing a crucial parameter in therapeutic decision-making. In vivo experiments corroborated the profound impact of protocol selection on tumor response. The fractionated regimen emerged as the standout performer, achieving a substantial reduction in tumor size over time, surpassing the efficacy of the non-fractionated approach. Additionally, the fractionated regimen exhibited efficacy also in targeting tumors in proximity but not in direct contact to the scaffolds. Our results address a critical gap in current research, highlighting the absence of a standardized protocol for optimizing the outcome of photodynamic therapy. The findings underscore the importance of personalized treatment strategies in achieving optimal therapeutic efficacy for precision cancer therapy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The therapeutic effect of mesenchymal stromal cells (MSCs) has been described for a variety of disorders, including those affecting musculoskeletal tissues. In this context, the literature reports several data about the regenerative effectiveness of MSCs derived from bone marrow, adipose tissue, and an amniotic membrane (BMSCs, ASCs, and hAMSCs, respectively), either when expanded or when acting as clinical-grade biologic pillars of products used at the point of care. To date, there is no evidence about the superiority of one source over the others from a clinical perspective. Therefore, a reliable characterization of the tissue-specific MSC types is mandatory to identify the most effective treatment, especially when tailored to the target disease. Because molecular characterization is a crucial parameter for cell definition, the need for reliable normalizers as housekeeping genes (HKGs) is essential. In this report, the stability levels of five commonly used HKGs ( ACTB , EF1A , GAPDH , RPLP0 , and TBP ) were sifted into BMSCs, ASCs, and hAMSCs. Adult and fetal/neonatal MSCs showed opposite HKG stability rankings. Moreover, by analyzing MSC types side-by-side, comparison-specific HKGs emerged. The effect of less performant HKG normalization was also demonstrated in genes coding for factors potentially involved in and predicting MSC therapeutic activity for osteoarthritis as a model musculoskeletal disorder, where the choice of the most appropriate normalizer had a higher impact on the donors rather than cell populations when compared side-by-side. In conclusion, this work confirms HKG source-specificity for MSCs and suggests the need for cell-type specific normalizers for cell source or condition-tailored gene expression studies.

Lung fibrosis is a complex process, with unknown underlying mechanisms, involving various triggers, diseases and stimuli. Different cell types (epithelial cells, endothelial cells, fibroblasts and macrophages) interact dynamically through multiple signalling pathways, including biochemical/molecular and mechanical signals, such as stiffness, affecting cell function and differentiation. Idiopathic pulmonary fibrosis (IPF) is the most common fibrosing interstitial lung disease (fILD), characterised by a notably high mortality. Unfortunately, effective treatments for advanced fILD, and especially IPF and non-IPF progressive fibrosing phenotype ILD, are still lacking. The development of pharmacological therapies faces challenges due to limited knowledge of fibrosis pathogenesis and the absence of pre-clinical models accurately representing the complex features of the disease. To address these challenges, new model systems have been developed to enhance the translatability of preclinical drug testing and bridge the gap to human clinical trials. The use of two- and three-dimensional in vitro cultures derived from healthy or diseased individuals allows for a better understanding of the underlying mechanisms responsible for lung fibrosis. Additionally, microfluidics systems, which replicate the respiratory system's physiology ex vivo , offer promising opportunities for the development of effective therapies, especially for IPF., Competing Interests: Conflict of interest: All authors have nothing to disclose., (Copyright ©The authors 2024.)

Background: The secretome of mesenchymal stromal cells isolated from the amniotic membrane (hAMSCs) has been extensively studied for its in vitro immunomodulatory activity as well as for the treatment of several preclinical models of immune-related disorders. The bioactive molecules within the hAMSCs secretome are capable of modulating the immune response and thus contribute to stimulating regenerative processes. At present, only a few studies have attempted to define the composition of the secretome, and several approaches, including multi-omics, are underway in an attempt to precisely define its composition and possibly identify key factors responsible for the therapeutic effect., Methods: In this study, we characterized the protein composition of the hAMSCs secretome by a filter-aided sample preparation (FASP) digestion and liquid chromatography-high resolution mass spectrometry (LC-MS) approach. Data were processed for gene ontology classification and functional protein interaction analysis by bioinformatics tools., Results: Proteomic analysis of the hAMSCs secretome resulted in the identification of 1521 total proteins, including 662 unique elements. A number of 157 elements, corresponding to 23.7%, were found as repeatedly characterizing the hAMSCs secretome, and those that resulted as significantly over-represented were involved in immunomodulation, hemostasis, development and remodeling of the extracellular matrix molecular pathways., Conclusions: Overall, our characterization enriches the landscape of hAMSCs with new information that could enable a better understanding of the mechanisms of action underlying the therapeutic efficacy of the hAMSCs secretome while also providing a basis for its therapeutic translation., (© 2023. The Author(s).)

Mesenchymal stromal cells (MSCs) are known for their immunomodulatory activity. Here, we report that MSCs isolated from the amniotic membrane of human term placenta (hAMSCs) impact CD8 T cell fate through a multifaceted mechanism. We observed that hAMSCs are able to impact the metabolism of naive CD8 lymphocytes by downregulating the phosphorylation of mTOR and AKT, thus blocking cell differentiation. This effect is due to the ability of hAMSCs to reduce the expression of two receptors, IL-12Rβ1 and IL-2RA, resulting in reduced phosphorylation of STAT4 and STAT5. In addition, hAMSCs reduce the expression of two transcriptional factors, Tbet and Eomes, directly involved in early effector cell commitment. Our results unravel an unknown feature of MSCs, offering alternative mechanistic insights into the effects of MSCs for the treatment of diseases characterized by an altered activation of memory subsets, such as autoimmune diseases and graft versus host disease., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Many preclinical studies have shown that birth-associated tissues, cells and their secreted factors, otherwise known as perinatal derivatives (PnD), possess various biological properties that make them suitable therapeutic candidates for the treatment of numerous pathological conditions. Nevertheless, in the field of PnD research, there is a lack of critical evaluation of the PnD standardization process: from preparation to in vitro testing, an issue that may ultimately delay clinical translation. In this paper, we present the PnD e-questionnaire developed to assess the current state of the art of methods used in the published literature for the procurement, isolation, culturing preservation and characterization of PnD in vitro . Furthermore, we also propose a consensus for the scientific community on the minimal criteria that should be reported to facilitate standardization, reproducibility and transparency of data in PnD research. Lastly, based on the data from the PnD e-questionnaire, we recommend to provide adequate information on the characterization of the PnD. The PnD e-questionnaire is now freely available to the scientific community in order to guide researchers on the minimal criteria that should be clearly reported in their manuscripts. This review is a collaborative effort from the COST SPRINT action (CA17116), which aims to guide future research to facilitate the translation of basic research findings on PnD into clinical practice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Janev, Banerjee, Weidinger, Dimec, Leskošek, Silini, Cirman, Wolbank, Ramuta, Jerman, Pandolfi, Di Pietro, Pozzobon, Giebel, Eissner, Ferk, Lang-Olip, Alviano, Soritau, Parolini and Kreft.)

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant epigenetic disorder with highly variable muscle involvement and disease progression. Ongoing clinical trials, aimed at counteracting muscle degeneration and disease progression in FSHD patients, increase the need for reliable biomarkers. Muscle magnetic resonance imaging (MRI) studies showed that the appearance of STIR-positive (STIR+) lesions in FSHD muscles represents an initial stage of muscle damage, preceding irreversible adipose changes. Our study aimed to investigate fibrosis, a parameter of muscle degeneration undetectable by MRI, in relation to disease activity and progression of FSHD muscles. We histologically evaluated collagen in FSHD1 patients' (STIR+ n = 27, STIR- n = 28) and healthy volunteers' (n = 12) muscles by picrosirius red staining. All patients (n = 55) performed muscle MRI before biopsy, 45 patients also after 1 year and 36 patients also after 2 years. Fat content (T1 signal) and oedema/inflammation (STIR signal) were evaluated at baseline and at 1- and 2-year MRI follow-up. STIR+ muscles showed significantly higher collagen compared to both STIR- (p = 0.001) and healthy muscles (p < 0.0001). STIR- muscles showed a higher collagen content compared to healthy muscles (p = 0.0194). FSHD muscles with a worsening in fatty infiltration during 1- (P = 0.007) and 2-year (P < 0.0001) MRI follow-up showed a collagen content of 3.6- and 3.7-fold higher compared to FSHD muscles with no sign of progression. Moreover, the fibrosis was significantly higher in STIR+ muscles who showed a worsening in fatty infiltration in a timeframe of 2 years compared to both STIR- (P = 0.0006) and STIR+ muscles with no sign of progression (P = 0.02). Fibrosis is a sign of muscle degeneration undetectable at MRI never deeply investigated in FSHD patients. Our data show that 23/27 of STIR+ and 12/28 STIR- muscles have a higher amount of collagen deposition compared to healthy muscles. Fibrosis is higher in FSHD muscles with a worsening in fatty infiltration thus suggesting that its evaluation with innovative non-invasive techniques could be a candidate prognostic biomarker for FSHD, to be used to stratify patients and to evaluate the efficacy of therapeutic treatments., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins' gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5'-3' untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes.

Multiple Sclerosis (MS) is a chronic inflammatory disease that affects the brain and spinal cord. Inflammation, demyelination, synaptic alteration, and neuronal loss are hallmarks detectable in MS. Experimental autoimmune encephalomyelitis (EAE) is an animal model widely used to study pathogenic aspects of MS. Autophagy is a process that maintains cell homeostasis by removing abnormal organelles and damaged proteins and is involved both in protective and detrimental effects that have been seen in a variety of human diseases, such as cancer, neurodegenerative diseases, inflammation, and metabolic disorders. This study is aimed at investigating the autophagy signaling pathway through the analysis of the main autophagic proteins including Beclin-1, microtubule-associated protein light chain (LC3, autophagosome marker), and p62 also called sequestosome1 (SQSTM1, substrate of autophagy-mediated degradation) in the hippocampus of EAE-affected mice. The expression levels of Beclin-1, LC3, and p62 and the Akt/mTOR pathway were examined by Western blot experiments. In EAE mice, compared to control animals, significant reductions of expression levels were detectable for Beclin-1 and LC3 II (indicating the reduction of autophagosomes), and p62 (suggesting that autophagic flux increased). In parallel, molecular analysis detected the deregulation of the Akt/mTOR signaling. Immunofluorescence double-labeling images showed co-localization of NeuN (neuronal nuclear marker) and Beclin-1, LC3, and p62 throughout the CA1 and CA3 hippocampal subfields. Taken together, these data demonstrate that activation of autophagy occurs in the neurons of the hippocampus in this experimental model., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Background: Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide. In addition to primary brain damage, systemic immune alterations occur, with evidence for dysregulated immune responses in aggravating TBI outcome and complications. However, immune dysfunction following TBI has been only partially understood, especially in the elderly who represent a substantial proportion of TBI patients and worst outcome. Therefore, we aimed to conduct an in-depth immunological characterization of TBI patients, by evaluating both adaptive (T and B lymphocytes) and innate (NK and monocytes) immune cells of peripheral blood mononuclear cells (PBMC) collected acutely (< 48 h) after TBI in young (18-45 yo) and elderly (> 65 yo) patients, compared to age-matched controls, and also the levels of inflammatory biomarkers., Results: Our data show that young respond differently than elderly to TBI, highlighting the immune unfavourable status of elderly compared to young patients. While in young only CD4 T lymphocytes are activated by TBI, in elderly both CD4 and CD8 T cells are affected, and are induced to differentiate into subtypes with low cytotoxic activity, such as central memory CD4 T cells and memory precursor effector CD8 T cells. Moreover, TBI enhances the frequency of subsets that have not been previously investigated in TBI, namely the double negative CD27- IgD- and CD38-CD24- B lymphocytes, and CD56dim CD16- NK cells, both in young and elderly patients. TBI reduces the production of pro-inflammatory cytokines TNF-α and IL-6, and the expression of HLA-DM, HLA-DR, CD86/B7-2 in monocytes, suggesting a compromised ability to drive a pro-inflammatory response and to efficiently act as antigen presenting cells., Conclusions: We described the acute immunological response induced by TBI and its relation with injury severity, which could contribute to pathologic evolution and possibly outcome. The focus on age-related immunological differences could help design specific therapeutic interventions based on patients' characteristics., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration. The genetic defect results in a reduced number of self-renewing muscle stem cells (MuSCs) and an impairment of their activation and differentiation, which lead to the exhaustion of skeletal muscle regeneration potential and muscle replacement by fibrotic and fatty tissue. In this study, we focused on an unexplored strategy to improve MuSC function and to preserve their niche based on the regenerative properties of mesenchymal stromal cells from the amniotic membrane (hAMSCs), that are multipotent cells recognized to have a role in tissue repair in different disease models. We demonstrate that the hAMSC secretome (CM hAMSC) and extracellular vesicles (EVs) isolated thereof directly stimulate the in vitro proliferation and differentiation of human myoblasts and mouse MuSC from dystrophic muscles. Furthermore, we demonstrate that hAMSC secreted factors modulate the muscle stem cell niche in dystrophic- mdx -mice. Interestingly, local injection of EV hAMSC in mdx muscles correlated with an increase in the number of activated Pax7+/Ki67+ MuSCs and in new fiber formation. EV hAMSCs also significantly reduced muscle collagen deposition, thus counteracting fibrosis and MuSCs exhaustion, two hallmarks of DMD. Herein for the first time we demonstrate that CM hAMSC and EVs derived thereof promote muscle regeneration by supporting proliferation and differentiation of resident muscle stem cells. These results pave the way for the development of a novel treatment to counteract DMD progression by reducing fibrosis and enhancing myogenesis in dystrophic muscles.

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.

The skeletal muscle has a very remarkable ability to regenerate upon injury under physiological conditions; however, this regenerative capacity is strongly diminished in physio-pathological conditions, such as those present in diseased or aged muscles. Many muscular dystrophies (MDs) are characterized by aberrant inflammation due to the deregulation of both the lymphoid and myeloid cell populations and the production of pro-inflammatory cytokines. Pathological inflammation is also observed in old muscles due to a systemic change in the immune system, known as "inflammaging". Immunomodulation represents, therefore, a promising therapeutic opportunity for different skeletal muscle conditions. However, the use of immunomodulatory drugs in the clinics presents several caveats, including their low stability in vivo, the need for high doses to obtain therapeutically relevant effects, and the presence of strong side effects. Within this context, the emerging field of nanomedicine provides the powerful tools needed to control the immune response. Nano-scale materials are currently being explored as biocarriers to release immunomodulatory agents in the damaged tissues, allowing therapeutic doses with limited off-target effects. In addition, the intrinsic immunomodulatory properties of some nanomaterials offer further opportunities for intervention that still need to be systematically explored. Here we exhaustively review the state-of-the-art regarding the use of nano-sized materials to modulate the aberrant immune response that characterizes some physio-pathological muscle conditions, such as MDs or sarcopenia (the age-dependent loss of muscle mass). Based on our learnings from cancer and immune tolerance induction, we also discuss further opportunities, challenges, and limitations of the emerging field of nano-immunomodulation.

The potential clinical applications of human amniotic membrane (hAM) and human amniotic epithelial cells (hAECs) in the field of regenerative medicine have been known in literature since long. However, it has yet to be elucidated whether hAM contains different anatomical regions with different plasticity and differentiation potential. Recently, for the first time, we highlighted many differences in terms of morphology, marker expression, and differentiation capabilities among four distinct anatomical regions of hAM, demonstrating peculiar functional features in hAEC populations. The aim of this study was to investigate in situ the ultrastructure of the four different regions of hAM by means of transmission electron microscopy (TEM) to deeply understand their peculiar characteristics and to investigate the presence and localization of secretory products because to our knowledge, there are no similar studies in the literature. The results of this study confirm our previous observations of hAM heterogeneity and highlight for the first time that hAM can produce extracellular vesicles (EVs) in a heterogeneous manner. These findings should be considered to increase efficiency of hAM applications within a therapeutic context.

Aim: The aetiology of cryptoglandular anal fistula (AF) is poorly understood. Evidence suggests that persistence and/or recurrence of the disease is more related to inflammatory than infectious factors. The aim of this study was to investigate the immune profile of cryptoglandular AF and to perform a histopathological characterization., Method: Fistulectomy was performed in all patients; healthy ischioanal fat from the same patients was used as a control. Samples were evaluated by the Luminex xMAP system for the detection of 27 analytes. AF tissues were analysed using immunofluorescence. Staining was performed using primary antibodies to identify M1 inflammatory and M2 anti-inflammatory macrophages. Selective staining of total T lymphocytes and different T lymphocyte subsets was performed., Results: Twenty patients with AF underwent a fistulectomy. Specific cytokine pathways differentiated AF from healthy tissue: pro-inflammatory cytokines interleukin (IL)-1β, IL-4, IL-8 and IL-17 and the anti-inflammatory cytokine IL-10 were overexpressed in AF compared with controls. Chemokines involved in macrophage recruitment (CCL2, CCL3, CCL4) were higher in AF than in healthy fatty tissue. Moreover, we showed that Tc17 cells characterize AF patients, thus confirming the enzyme-linked immunosorbent assay data. Furthermore, elevated infiltration of CD68+ myeloid cells and a reduction of the M1/M2 ratio characterize AF patients., Conclusion: A combination of inflammatory cytokines, chemokines and growth factors reside in the wound microenvironment of AF patients. For the first time an important prevalence of Tc17 cells and a reduction in the M1/M2 ratio was observed, thus suggesting new insights into the immunological characterization of AF patients., (© 2022 The Authors. Colorectal Disease published by John Wiley & Sons Ltd on behalf of Association of Coloproctology of Great Britain and Ireland.)

Perinatal derivatives or PnDs refer to tissues, cells and secretomes from perinatal, or birth-associated tissues. In the past 2 decades PnDs have been highly investigated for their multimodal mechanisms of action that have been exploited in various disease settings, including in different cancers and infections. Indeed, there is growing evidence that PnDs possess anticancer and antimicrobial activities, but an urgent issue that needs to be addressed is the reproducible evaluation of efficacy, both in vitro and in vivo . Herein we present the most commonly used functional assays for the assessment of antitumor and antimicrobial properties of PnDs, and we discuss their advantages and disadvantages in assessing the functionality. This review is part of a quadrinomial series on functional assays for the validation of PnDs spanning biological functions such as immunomodulation, anticancer and antimicrobial, wound healing, and regeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Silini, Ramuta, Pires, Banerjee, Dubus, Gindraux, Kerdjoudj, Maciulatis, Weidinger, Wolbank, Eissner, Giebel, Pozzobon, Parolini and Kreft.)

Muscle-resident non-myogenic mesenchymal cells play key roles that drive successful tissue regeneration within the skeletal muscle stem cell niche. These cells have recently emerged as remarkable therapeutic targets for neuromuscular disorders, although to date they have been poorly investigated in facioscapulohumeral muscular dystrophy (FSHD). In this study, we characterised the non-myogenic mesenchymal stromal cell population in FSHD patients' muscles with signs of disease activity, identified by muscle magnetic resonance imaging (MRI), and compared them with those obtained from apparently normal muscles of FSHD patients and from muscles of healthy, age-matched controls. Our results showed that patient-derived cells displayed a distinctive expression pattern of mesenchymal markers, along with an impaired capacity to differentiate towards mature adipocytes in vitro, compared with control cells. We also demonstrated a significant expansion of non-myogenic mesenchymal cells (identified as CD201- or PDGFRA-expressing cells) in FSHD muscles with signs of disease activity, which correlated with the extent of intramuscular fibrosis. In addition, the accumulation of non-myogenic mesenchymal cells was higher in FSHD muscles that deteriorate more rapidly. Our results prompt a direct association between an accumulation, as well as an altered differentiation, of non-myogenic mesenchymal cells with muscle degeneration in FSHD patients. Elucidating the mechanisms and cellular interactions that are altered in the affected muscles of FSHD patients could be instrumental to clarify disease pathogenesis and identifying reliable novel therapeutic targets., (© 2022. The Author(s).)

Perinatal tissues, mainly the placenta and umbilical cord, contain a variety of different somatic stem and progenitor cell types, including those of the hematopoietic system, multipotent mesenchymal stromal cells (MSCs), epithelial cells and amnion epithelial cells. Several of these perinatal derivatives (PnDs), as well as their secreted products, have been reported to exert immunomodulatory therapeutic and regenerative functions in a variety of pre-clinical disease models. Following experience with MSCs and their extracellular vesicle (EV) products, successful clinical translation of PnDs will require robust functional assays that are predictive for the relevant therapeutic potency. Using the examples of T cell and monocyte/macrophage assays, we here discuss several assay relevant parameters for assessing the immunomodulatory activities of PnDs. Furthermore, we highlight the need to correlate the in vitro assay results with preclinical or clinical outcomes in order to ensure valid predictions about the in vivo potency of therapeutic PnD cells/products in individual disease settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Papait, Silini, Gazouli, Malvicini, Muraca, O’Driscoll, Pacienza, Toh, Yannarelli, Ponsaerts, Parolini, Eissner, Pozzobon, Lim and Giebel.)

Amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties demonstrated in vitro and in vivo in various diseases in which the dysregulated immune system plays a major role. The immunomodulatory and pro-regenerative effects of MSCs, among which hAMSCs lie in the bioactive factors they secrete and in their paracrine activity, is well known. The mix of these factors (i.e., secretome) can be either freely secreted or conveyed by extracellular vesicles (EV), thus identifying two components in the cell secretome: EV-free and EV fractions. This study aimed to discern the relative impact of the individual components on the immunomodulatory action of the hAMSC secretome in order to obtain useful information for implementing future therapeutic approaches using immunomodulatory therapies based on the MSC secretome. To this aim, we isolated EVs from the hAMSC secretome (hAMSC-CM) by ultracentrifugation and validated the vesicular product according to the International Society for Extracellular Vesicles (ISEV) criteria. EVs were re-diluted in serum-free medium to maintain the EV concentration initially present in the original CM. We compared the effects of the EV-free and EV fractions with those exerted by hAMSC-CM in toto on the activation and differentiation of immune cell subpopulations belonging to both the innate and adaptive immune systems. We observed that the EV-free fraction, similar to hAMSC-CM in toto , a) decreases the proliferation of activated peripheral blood mononuclear cells (PBMC), b) reduces the polarization of T cells toward inflammatory Th subsets, and induces the induction of regulatory T cells; c) affects monocyte polarization to antigen-presenting cells fostering the acquisition of anti-inflammatory macrophage (M2) markers; and d) reduces the activation of B lymphocytes and their maturation to plasma cells. We observed instead that all investigated EV fractions, when used in the original concentrations, failed to exert any immunomodulatory effect, even though we show that EVs are internalized by various immune cells within PBMC. These findings suggest that the active component able to induce immune regulation, tested at original concentrations, of the hAMSC secretome resides in factors not conveyed in EVs. However, EVs isolated from hAMSC could exert actions on other cell types, as reported by others., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Papait, Ragni, Cargnoni, Vertua, Romele, Masserdotti, Perucca Orfei, Signoroni, Magatti, Silini, De Girolamo and Parolini.)

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Flores, Pipino, Jerman, Liarte, Gindraux, Kreft, Nicolas, Pandolfi, Tratnjek, Giebel, Pozzobon, Silini, Parolini, Eissner and Lang-Olip.)

The tumor microenvironment (TME) is comprised of different cellular components, such as immune and stromal cells, which co-operate in unison to promote tumor progression and metastasis. In the last decade, there has been an increasing focus on one specific component of the TME, the stromal component, often referred to as Cancer-Associated Fibroblasts (CAF). CAF modulate the immune response and alter the composition of the extracellular matrix with a decisive impact on the response to immunotherapies and conventional chemotherapy. The most recent publications based on single-cell analysis have underlined CAF heterogeneity and the unique plasticity that strongly impact the TME. In this review, we focus not only on the characterization of CAF based on the most recent findings, but also on their impact on the immune system. We also discuss clinical trials and preclinical studies where targeting CAF revealed controversial results. Therefore, future efforts should focus on understanding the functional properties of individual subtypes of CAF, taking into consideration the peculiarities of each pathological context.

Muscular dystrophies are a group of rare genetic disorders characterized by progressive muscle weakness, which, in the most severe forms, leads to the patient's death due to cardiorespiratory problems. There is still no cure available for these diseases and significant effort is being placed into developing new strategies to either correct the genetic defect or to compensate muscle loss by stimulating skeletal muscle regeneration. However, the vast anatomical extension of the target tissue poses great challenges to these goals, highlighting the need for complementary strategies. Nanomedicine is an actively evolving field that merges nanotechnologies with biomedical and pharmaceutical sciences. It holds great potential in regenerative medicine, both in supporting tissue engineering and regeneration, and in optimizing drug and oligonucleotide delivery and gene therapy strategies. In this review, we will summarize the state-of-the-art in the field of nanomedicine applied to skeletal muscle regeneration. We will discuss the recent work toward the development of nanopatterned scaffolds for tissue engineering, the efforts in the synthesis of organic and inorganic nanoparticles for gene therapy and drug delivery applications, as well as their use as immune modulators. Although nanomedicine holds great promise for muscle and other degenerative diseases, many challenges still need to be systematically addressed to assure a smooth transition from the bench to the bedside. This article is categorized under: Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement., (© 2022 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals LLC.)

Background: The COVID-19 pandemic affected healthcare systems worldwide. Predictive models developed by Artificial Intelligence (AI) and based on timely, centralized and standardized real world patient data could improve management of COVID-19 to achieve better clinical outcomes. The objectives of this manuscript are to describe the structure and technologies used to construct a COVID-19 Data Mart architecture and to present how a large hospital has tackled the challenge of supporting daily management of COVID-19 pandemic emergency, by creating a strong retrospective knowledge base, a real time environment and integrated information dashboard for daily practice and early identification of critical condition at patient level. This framework is also used as an informative, continuously enriched data lake, which is a base for several on-going predictive studies., Methods: The information technology framework for clinical practice and research was described. It was developed using SAS Institute software analytics tool and SAS® Vyia® environment and Open-Source environment R ® and Python ® for fast prototyping and modeling. The included variables and the source extraction procedures were presented., Results: The Data Mart covers a retrospective cohort of 5528 patients with SARS-CoV-2 infection. People who died were older, had more comorbidities, reported more frequently dyspnea at onset, had higher d-dimer, C-reactive protein and urea nitrogen. The dashboard was developed to support the management of COVID-19 patients at three levels: hospital, single ward and individual care level., Interpretation: The COVID-19 Data Mart based on integration of a large collection of clinical data and an AI-based integrated framework has been developed, based on a set of automated procedures for data mining and retrieval, transformation and integration, and has been embedded in the clinical practice to help managing daily care. Benefits from the availability of a Data Mart include the opportunity to build predictive models with a machine learning approach to identify undescribed clinical phenotypes and to foster hospital networks. A real-time updated dashboard built from the Data Mart may represent a valid tool for a better knowledge of epidemiological and clinical features of COVID-19, especially when multiple waves are observed, as well as for epidemic and pandemic events of the same nature (e. g. with critical clinical conditions leading to severe pulmonary inflammation). Therefore, we believe the approach presented in this paper may find several applications in comparable situations even at region or state levels. Finally, models predicting the course of future waves or new pandemics could largely benefit from network of DataMarts., Competing Interests: Declarations of Competing Interest None., (Copyright © 2022. Published by Elsevier B.V.)

Aims: The aim of the HIPGEN consortium is to develop the first cell therapy product for hip fracture patients using PLacental-eXpanded (PLX-PAD) stromal cells., Methods: HIPGEN is a multicentre, multinational, randomized, double-blind, placebo-controlled trial. A total of 240 patients aged 60 to 90 years with low-energy femoral neck fractures (FNF) will be allocated to two arms and receive an intramuscular injection of either 150 × 10 6 PLX-PAD cells or placebo into the medial gluteal muscle after direct lateral implantation of total or hemi hip arthroplasty. Patients will be followed for two years. The primary endpoint is the Short Physical Performance Battery (SPPB) at week 26. Secondary and exploratory endpoints include morphological parameters (lean body mass), functional parameters (abduction and handgrip strength, symmetry in gait, weightbearing), all-cause mortality rate and patient-reported outcome measures (Lower Limb Measure, EuroQol five-dimension questionnaire). Immunological biomarker and in vitro studies will be performed to analyze the PLX-PAD mechanism of action. A sample size of 240 subjects was calculated providing 88% power for the detection of a 1 SPPB point treatment effect for a two-sided test with an α level of 5%., Conclusion: The HIPGEN study assesses the efficacy, safety, and tolerability of intramuscular PLX-PAD administration for the treatment of muscle injury following arthroplasty for hip fracture. It is the first phase III study to investigate the effect of an allogeneic cell therapy on improved mobilization after hip fracture, an aspect which is in sore need of addressing for the improvement in standard of care treatment for patients with FNF. Cite this article: Bone Jt Open  2022;3(4):340-347.

Cancer spheroids are in vitro 3D models that became crucial in nanomaterials science thanks to the possibility of performing high throughput screening of nanoparticles and combined nanoparticle-drug therapies on in vitro models. However, most of the current spheroid analysis methods involve manual steps. This is a time-consuming process and is extremely liable to the variability of individual operators. For this reason, rapid, user-friendly, ready-to-use, high-throughput image analysis software is necessary. In this work, we report the INSIDIA 2.0 macro, which offers researchers high-throughput and high content quantitative analysis of in vitro 3D cancer cell spheroids and allows advanced parametrization of the expanding and invading cancer cellular mass. INSIDIA has been implemented to provide in-depth morphologic analysis and has been used for the analysis of the effect of graphene quantum dots photothermal therapy on glioblastoma (U87) and pancreatic cancer (PANC-1) spheroids. Thanks to INSIDIA 2.0 analysis, two types of effects have been observed: In U87 spheroids, death is accompanied by a decrease in area of the entire spheroid, with a decrease in entropy due to the generation of a high uniform density spheroid core. On the other hand, PANC-1 spheroids' death caused by nanoparticle photothermal disruption is accompanied with an overall increase in area and entropy due to the progressive loss of integrity and increase in variability of spheroid texture. We have summarized these effects in a quantitative parameter of spheroid disruption demonstrating that INSIDIA 2.0 multiparametric analysis can be used to quantify cell death in a non-invasive, fast, and high-throughput fashion.

Fracture non-union is a challenging orthopaedic issue and a socio-economic global burden. Several biological therapies have been introduced to improve traditional surgical approaches. Among these, the latest research has been focusing on adipose tissue as a powerful source of mesenchymal stromal cells, namely, adipose-derived stem cells (ADSCs). ADSC are commonly isolated from the stromal vascular fraction (SVF) of liposuctioned hypodermal adipose tissue, and their applications have been widely investigated in many fields, including non-union fractures among musculoskeletal disorders. This review aims at providing a comprehensive update of the literature on clinical application of ADSCs for the treatment of non-unions in humans. The study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Only three articles met our inclusion criteria, with a total of 12 cases analyzed for demographics and harvesting, potential manufacturing and implantation of ADSCs. The review of the literature suggests that adipose derived cell therapy can represent a promising alternative in bone regenerative medicine for the enhancement of non-unions and bone defects. The low number of manuscripts reporting ADSC-based therapies for long bone fracture healing suggests some critical issues that are discussed in this review. Nevertheless, further investigations on human ADSC therapies are needed to improve the knowledge on their translational potential and to possibly achieve a consensus on their use for such applications.

Background: Implantation of the amniotic membrane and their derivatives can have a beneficial effect on tissue repair and regeneration. We report for the first time the implant of an amniotic membrane in a patient affected by cryptoglandular anal fistula., Methods: A patch of human amniotic membrane was implanted in a female patient affected by an anterior transphincteric fistula. Following an accurate curettage of the anal fistula, the cryopreserved amniotic membrane was thawed and then washed in the operating room; one side of the membrane was transfixed with a resorbable suture thus creating an implantable fusiform patch. The membrane was subsequently implanted into the fistula tract from the external to the internal opening. The inner and outer parts of the membrane were then sutured to the internal and external fistula openings., Results: No intraoperative or postoperative complications occurred. The patient was discharged one day after the procedure after an uneventful hospitalization. At the 1-week, 1- and 3-month follow-up visits no pain (VAS 0) was referred by the patient and no inflammation was evident at the level of the previous external fistula opening., Conclusions: The implant of human amniotic membrane in a patient affected by cryptoglandular anal fistula was safely and easily performed. Moreover, future studies to assess the efficacy in the long-term follow-up are needed.

There are controversial results about the role of "ex novo" HLA-DR expression by tumor cells and its correlation with the oncological outcomes. Unfortunately, little is known about HLA-DR expression in laryngeal cancer tumor cells. The main purpose of this retrospective study is to strengthen the usefulness of studying "ex novo" HLA-DR expression on tumor cells from primary laryngeal squamous cell carcinoma (LSCC) patients and investigate its correlation with clinical outcome. We analyzed HLA-DR expression by immunohistochemical analysis in 56 patients with LSCC. The "ex novo" HLA-DR expression on laryngeal cancer tumor cells, assessing non-neoplastic LSCC - adjacent tissue, and the association of HLA-DR expression (HLA-DR+) with clinical outcomes were investigated. HLA-DR+ tumor cells were detected in 18/56 LSCC patients (32.1%). All specimens of non-neoplastic laryngeal carcinoma-adjacent tissue resulted HLA-DR negative (HLA-DR-). A statistically significant association was observed between HLA-DR + and well differentiated tumors (G1) (p<0.001). The Kaplan-Meier method showed how HLA-DR+ is significantly associated with both a better disease specific survival (HLA-DR+=100% vs. HLA-DR-=77.4%; p=0.047) and a better relapse free survival (HLA-DR+=100% vs. HLA-DR-=72.3%; p=0.021). Cox regression univariate analysis for death of disease confirmed a higher HR for HLA-DR absence on the surface of epithelial tumor cell [HR:37.489; 95% CI:0.750-18730.776; p=0.253] and for high-grade (G3) tumors [HR:18.601; 95% CI:3.613-95.764; p<0.0001]. Our results confirm that MHC class II HLA-DR expression is activated in a sub-set of LSCC patients. Evaluation of HLA-DR expression in LSCC could be useful for prognosis and future approaches towards personalized therapy., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)