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An abdominal aortic aneurysm (AAA) is described as a gradual and localized permanent expansion of the aorta resulting from the weakening of the vascular wall. The key aspects of AAA's progression are high proteolysis of the structural elements of the vascular wall, the depletion of vascular smooth muscle cells (VSMCs), and a chronic immunoinflammatory response. The pathological mechanisms underpinning the development of an AAA are complex and still unknown. At present, there are no successful drug treatments available that can slow the progression of an AAA or prevent the rupture of the aneurysmal vascular wall. Recently, it has been suggested that endothelial cellular senescence may be involved in vascular aging and vascular aging diseases, but there is no clear correlation between cellular senescence and AAAs. Therefore, the aim of this study was to identify the presence of senescent cells on the vascular wall of aneurysmatic abdominal aortas and to correlate their distribution with the morphological markers of AAAs. Pathological and healthy segments of abdominal aortas were collected during repair surgery and immediately processed for histological and immunohistochemical analyses. Hematoxylin/eosin, Verhoeff–van Gieson, and Goldner's Masson trichrome staining procedures were carried out to investigate the morphological features related to the pathology. Immunohistochemical investigations for the p21cip1/waf1, p53, and NFkB markers were carried out to selectively identify positive cells in the vascular wall of the AAA samples related to cellular senescence and an inflammatory microenvironment. The results revealed the presence of a few senescent vascular cells on the aneurysmatic wall of the abdominal aortas, surrounded by a highly inflamed microenvironment that was highly expressed in the tunica media and adventitia of both pathological and healthy segments. Our data demonstrate the presence of senescent vascular cells in AAA samples, which could enhance the promotion of a high inflammatory vascular microenvironment, supporting the evolution of the pathology. Although this study was based on only two cases, the results highlight the importance of targeting cellular senescence to reduce an inflammatory microenvironment, which can support the progression of age-related diseases. [ABSTRACT FROM AUTHOR]

Teucrium chamaedrys L. is a typical European–Mediterranean species of the genus Teucrium. Among the phenolic compounds belonging to phenylethanoid glycosides (PGs), teucrioside (TS) is only found in this species, and it was previously demonstrated to be produced by in vitro-elicited cell cultures at levels higher than those found in leaves. However, T. chamaedrys cell suspension extracts (Cell-Ex) and pure TS have not been investigated yet for any biological effects. In this study, we evaluated the antioxidant and anti-melanogenesis activity of both Cell-Ex and TS in B16-F10 mouse melanoma cells. The results showed that Cell-Ex inhibited the reactive oxygen species formation evoked in B16-F10 cells by tert-butyl hydroperoxide and 5 J/cm2 of UVA, as well as the melanin increase stimulated by α-MSH or 20 J/cm2 of UVA. In parallel, a TS concentration equivalent to that present in Cell-Ex recorded the same biological effect profile, suggesting the main contribution of TS to the antioxidant and anti-melanogenic properties of Cell-Ex. Both Cell-Ex and TS also modulated the melanogenesis pathway through their ability to inhibit the tyrosinase activity both in a cell-free system and in B16-F10 cells stimulated by α-MSH. These results support the potential cosmeceutical use of Cell-Ex for protection against photooxidative damage and hyperpigmentation. [ABSTRACT FROM AUTHOR]

The most common pulmonary findings in opiate-related fatalities are congestion and oedema, as well as acute and/or chronic alveolar haemorrhage, the cause of which is thought to be a damage to the capillary endothelium related to ischemia. Human vascular mesenchymal stromal cells (vMSCs) play a fundamental role in tissue regeneration and repair after endothelial cell injury, and they express opioid receptors. The aim of this study was to assess the effect of in vitro morphine exposure on the physiological activity and maintenance of human vMSCs. vMSCs were obtained from abdominal aorta fragments collected during surgery repair and were exposed to incremental doses (0.1 mM, 0.4 mM, 0.8 mM and 1 mM) of morphine sulphate for 7 days. The effect was investigated through cell viability assessment, proliferation assay, reactive oxygen species (ROS) detection assay, senescence-associated β-galactosidase assay, senescent-related markers (p21WAF1/CIP1 and p16INK4) and the apoptosis-related marker caspase 3. Moreover, an ultrastructural analysis by transmission electron microscopy and in vitro vascular differentiation were evaluated. Results showed a decrease of the cellular metabolic activity, a pro-oxidant and pro-senescence effect, an increase in intracellular ROS and the activation of the apoptosis signalling, as well as ultrastructural modifications and impairment of vascular differentiation after morphine treatment of vMSC. Although confirmation studies are required on real fatal opiate intoxications, the approach based on morphological and immunofluorescence methodologies may have a high potential also as a useful tool or as a complementary method in forensic pathology. The application of these techniques in the future may lead to the identification of new markers and morphological parameters useful as complementary investigations for drug-related deaths. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we aim to characterize and compare synovial fluid MSCs (sf-MSCs), isolated from OA joints, with healthy sf-MSCs, investigating the senescence hallmarks and how this state could affect cartilage repair. Sf-MSCs were isolated from tibiotarsal joints of healthy and diseased horses with an established diagnosis of OA with an age ranging from 8 to 14 years. Cells were cultured in vitro and characterized for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and the expression of senescent markers. To evaluate the influence of senescence on chondrogenic differentiation, OA sf-MSCs were stimulated in vitro for up to 21 days with chondrogenic factors, and the expression of chondrogenic markers was compared with healthy sf-MSCs. Our findings demonstrated the presence of senescent sf-MSCs in OA joints with impaired chondrogenic differentiation abilities, which could have a potential influence on OA progression. [ABSTRACT FROM AUTHOR]

Simple Summary: Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer, is mostly associated with smoking and has a low survival rate. Among the different alterations in genes identified in SCLC, those related to the MYC family have emerged as highly relevant, alterations MYCN particularly define an aggressive and immunotherapy resistant SCLC subgroup. Due to its highly restricted expression in normal cells, MYCN is an ideal target for precision medicine, though it is difficult to target with traditional approaches. We propose an innovative approach to target MYCN in SCLC by an MYCN-specific expression inhibition at the level of gene transcription through an antigene oligonucleotide (BGA002). BGA002 exerted a potent and specific MYCN silencing in MYCN-expressing SCLC, leading to reversion of specific pathways and induction of cell death. Moreover, systemic administration of BGA002 inhibited tumor progression and significantly increased survival in SCLC mouse models, while also overcoming multidrug resistance. Small-cell lung cancer (SCLC) is the most aggressive lung cancer type, and is associated with smoking, low survival rate due to high vascularization, metastasis and drug resistance. Alterations in MYC family members are biomarkers of poor prognosis for a large number of SCLC. In particular, MYCN alterations define SCLC cases with immunotherapy failure. MYCN has a highly restricted pattern of expression in normal cells and is an ideal target for cancer therapy but is undruggable by traditional approaches. We propose an innovative approach to MYCN inhibition by an MYCN-specific antigene—PNA oligonucleotide (BGA002)—as a new precision medicine for MYCN-related SCLC. We found that BGA002 profoundly and specifically inhibited MYCN expression in SCLC cells, leading to cell-growth inhibition and apoptosis, while also overcoming multidrug resistance. These effects are driven by mTOR pathway block in concomitance with autophagy reactivation, thus avoiding the side effects of targeting mTOR in healthy cells. Moreover, we identified an MYCN-related SCLC gene signature comprehending CNTFR, DLX5 and TNFAIP3, that was reverted by BGA002. Finally, systemic treatment with BGA002 significantly increased survival in MYCN-amplified SCLC mouse models, including in a multidrug-resistant model in which tumor vascularization was also eliminated. These findings warrant the clinical testing of BGA002 in MYCN-related SCLC. [ABSTRACT FROM AUTHOR]

The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs. [ABSTRACT FROM AUTHOR]

Autosomal-dominant leukodystrophy (ADLD) is a rare fatal neurodegenerative disorder with overexpression of the nuclear lamina component, Lamin B1 due to LMNB1 gene duplication or deletions upstream of the gene. The molecular mechanisms responsible for driving the onset and development of this pathology are not clear yet. Vacuolar demyelination seems to be one of the most significant histopathological observations of ADLD. Considering the role of oligodendrocytes, astrocytes, and leukemia inhibitory factor (LIF)-activated signaling pathways in the myelination processes, this work aims to analyze the specific alterations in different cell populations from patients with LMNB1 duplications and engineered cellular models overexpressing Lamin B1 protein. Our results point out, for the first time, that astrocytes may be pivotal in the evolution of the disease. Indeed, cells from ADLD patients and astrocytes overexpressing LMNB1 show severe ultrastructural nuclear alterations, not present in oligodendrocytes overexpressing LMNB1. Moreover, the accumulation of Lamin B1 in astrocytes induces a reduction in LIF and in LIF-Receptor (LIF-R) levels with a consequential decrease in LIF secretion. Therefore, in both our cellular models, Jak/Stat3 and PI3K/Akt axes, downstream of LIF/LIF-R, are downregulated. Significantly, the administration of exogenous LIF can partially reverse the toxic effects induced by Lamin B1 accumulation with differences between astrocytes and oligodendrocytes, highlighting that LMNB1 overexpression drastically affects astrocytic function reducing their fundamental support to oligodendrocytes in the myelination process. In addition, inflammation has also been investigated, showing an increased activation in ADLD patients' cells. [ABSTRACT FROM AUTHOR]

Purpose: To shed light on the idea that mesenchymal stem/stromal cells (MSCs) recruited in synovium (SM) (i.e.Synovium-Derived Stromal Cells, SDSCs) could be involved in Osteoarthritis (OA) pathophysiology. Attention was also paid to a further stromal cell type with a peculiar ultrastructure called telocytes (TCs), whose role is far from clarified. Methods: In the present in vitro study, we compared SDSCs isolated from healthy and OA subjects in terms of phenotype, morphology and differentiation potential as well as in their capability to activate normal Peripheral Blood Mononuclear Cells (PBMCs). Histological, immunohistochemical and ultrastructural analyses were integrated by qRT-PCR and functional resorbing assays. Results: Our data demonstrated that both SDSC populations stimulated the formation of osteoclasts from PBMCs: the osteoclast-like cells generated by healthy-SDSCs via transwell co-cultures were inactive, while OA-derived SDSCs have a much greater effectiveness. Moreover, the presence of TCs was more evident in cultures obtained from OA subjects and suggests a possible involvement of these cells in OA. Conclusions: Osteoclastogenic differentiation capability of PBMCs from OA subjects, also induced by B synoviocytes has been already documented. Here we hypothesized that SDSCs, generally considered for their regenerative potential in cartilage lesions, have also a role in the onset/maintenance of OA. Clinical relevance: Our observations may represent an interesting opportunity for the development of a holistic approach for OA treatment, that considers the multifaceted capability of MSCs in relation to the environment. [ABSTRACT FROM AUTHOR]

Senescence can impair the therapeutic potential of stem cells. In this study, senescence-associated morphofunctional changes in periosteum-derived progenitor cells (PDPCs) from old and young individuals were investigated by combining cytofluorimetry, immunohistochemistry, and transmission electron microscopy. Cell cycle analysis demonstrated a large number of G0/G1 phase cells in PDPCs from old subjects and a progressive accumulation of G0/G1 cells during passaging in cultures from young subjects. Cytofluorimetry documented significant changes in light scattering parameters and closely correlated with the ultrastructural features, especially changes in mitochondrial shape and autophagy, which are consistent with the mitochondrial-lysosomal axis theory of ageing. The combined morphological, biofunctional, and ultrastructural approach enhanced the flow cytometric study of PDPC ageing. We speculate that impaired autophagy, documented in replicative senescent and old PDPCs, reflect a switch from quiescence to senescence. Its demonstration in a tissue with limited turnover—like the cambium layer of the periosteum, where reversible quiescence is the normal stem cell state throughout life—adds a new piece to the regenerative medicine jigsaw in an ageing society. [ABSTRACT FROM AUTHOR]

Wharton’s jelly (WJ) is an important source of mesenchymal stem cells (MSCs) both in human and other animals. The aim of this study was to compare human and equine WJMSCs. Human and equine WJMSCs were isolated and cultured using the same protocols and culture media. Cells were characterized by analysing morphology, growth rate, migration and adhesion capability, immunophenotype, differentiation potential and ultrastructure. Results showed that human and equine WJMSCs have similar ultrastructural details connected with intense synthetic and metabolic activity, but differ in growth, migration, adhesion capability and differentiation potential. In fact, at the scratch assay and transwell migration assay, the migration ability of human WJMSCs was higher (P < 0.05) than that of equine cells, while the volume of spheroids obtained after 48 h of culture in hanging drop was larger than the volume of equine ones (P < 0.05), demonstrating a lower cell adhesion ability. This can also revealed in the lower doubling time of equine cells (3.5 ± 2.4 days) as compared to human (6.5 ± 4.3 days) (P < 0.05), and subsequently in the higher number of cell doubling after 44 days of culture observed for the equine (20.3 ± 1.7) as compared to human cells (8.7 ± 2.4) (P < 0.05), and to the higher (P < 0.05) ability to form fibroblast colonies at P3. Even if in both species tri-lineage differentiation was achieved, equine cells showed an higher chondrogenic and osteogenic differentiation ability (P < 0.05). Our findings indicate that, although the ultrastructure demonstrated a staminal phenotype in human and equine WJMSCs, they showed different properties reflecting the different sources of MSCs. [ABSTRACT FROM AUTHOR]

Abstract: Estimating the post mortem interval (PMI) is still a crucial step in Forensic Pathology. Although several methods are available for assessing the PMI, a precise estimation is still quite unreliable and can be inaccurate. The present study aimed to investigate the immunohistochemical distribution and mRNA expression of hypoxia inducible factor (HIF‐1α) in post mortem gingival tissues to establish a correlation between the presence of HIF‐1α and the time since death, with the final goal of achieving a more accurate PMI estimation. Samples of gingival tissues were obtained from 10 cadavers at different PMIs (1–3 days, 4–5 days and 8–9 days), and were processed for immunohistochemistry and quantitative reverse transcription‐polymerase chain reaction. The results showed a time‐dependent correlation of HIF‐1α protein and its mRNA with different times since death, which suggests that HIF‐1α is a potential marker for PMI estimation. The results showed a high HIF‐1α protein signal that was mainly localized in the stratum basale of the oral mucosa in samples collected at a short PMI (1–3 days). It gradually decreased in samples collected at a medium PMI (4–5 days), but it was not detected in samples collected at a long PMI (8–9 days). These results are in agreement with the mRNA data. These data indicate an interesting potential utility of Forensic Anatomy‐based techniques, such as immunohistochemistry, as important complementary tools to be used in forensic investigations. [ABSTRACT FROM AUTHOR]

Adult stem cells are a promising cell source for cartilage regeneration. They resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Cobalt chloride (CoCl2) imitates hypoxia in vitro by stabilizing hypoxia-inducible factor-alpha (HIF-1α), which is the master regulator in the cellular adaptive response to hypoxia. In this study, the influence of CoCl2 on the chondrogenic potential of human MSCs, isolated from dental pulp, umbilical cord, and adipose tissue, was investigated. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 μM. Cell viability, HIF-1α protein synthesis, and the expression of the chondrogenic markers were analyzed. The results showed that the CoCl2 supplementation had no effect on cell viability, while the upregulation of chondrogenic markers such as SOX9, COL2A1, VCAN, and ACAN was dependent on the cellular source. This study shows that hypoxia, induced by CoCl2 treatment, can differently influence the behavior of MSCs, isolated from different sources, in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies. [ABSTRACT FROM AUTHOR]

Purpose: Collagen Type 1 and Non-Collagenous Proteins (NCPs) are fundamental elements of the human dentin organic matrix. Among the NCPs Osteopontin (OPN), Bone SialoProtein (BSP), Dentin Matrix Protein 1 (DMP1), Dentin Sialo PhosfoProtein (DSPP) are the most represented. These acidic proteins are produced by odontoblasts and their role is essential during mineralization process. Recent studies have evaluated how the application of NCPs, or their analogs, added in the dental materials promote the process of re-mineralization in both interfibrillar and intrafibrillar space. The intrafibrillar re-mineralization process is fundamental for a long-lasting adhesive layer because it prevents the collagen fibers from the degradation by the metalloproteinases (MMPs). DMP1 and the DSPP have been already evaluated, but few researches have focused on BSP, especially its expression in human carious dentin. The objective of this study was to compare, by histomorphometric analysis, the different pattern and distribution of BSP in the human carious dentin vs sound dentin. Materials and Methods: Ten decayed teeth -- test group -- and ten sound teeth -- control group -- were processed and subsequently submitted to the immunolabeling process through BSP-sensitive antibody. Results: In the control samples BSP protein was not marked, except in the predentinal layer. In the test group; however, the distribution pattern of the BSP protein was statistically significant both in the sclerotic dentin layer, underlying the carious lesion and in the reaction dentin layer. The intensity of labeling decreased from the lesion towards the pulp and then intensified in the reaction dentin front. Conclusion: The present study gives new considerations on the presence and distribution of BSP in carious dentin, indicating how the protein is mostly expressed in the sclerotic and reaction dentin front. This pattern distribution is therefore compatible with a "self-remineralizing" response by dentin matrix in response to carious stimuli. [ABSTRACT FROM AUTHOR]

Purpose: We describe a novel class of antitumor amphiphilic amines (RCn) based on a tricyclic amine hydrophilic head and a hydrophobic linear alkyl tail of variable length. Methods: We tested the lead compound, RC16, for cytotoxicity and mechanism of cell death in several cancer cell lines, anti tumor efficacy in mouse tumor models, and ability to encapsulate chemotherapy drugs. Results: These compounds displayed strong cytotoxic activity against cell lines derived from both pediatric and adult cancers. The IC50 of the lead compound, RC16, for normal cells including human keratinocytes, human fibroblasts and human umbilical vein endothelial cells was tenfold higher than for tumor cells. RC16 exhibited significant antitumor effects in vivo using several human xenografts and a metastatic model of murine neuroblastoma by both intravenous and oral administration routes. The amphiphilic character of RC16 triggered a spontaneous molecular self-assembling in water with formation of micelles allowing complexation of Doxorubicin, Etoposide and Paclitaxel. These micelles significantly improved the in vitro antitumor activity of these drugs as the enhancement of their aqueous solubility also improved their biologic availability. Conclusions: RC16 and related amphiphilic amines may be useful as a novel cancer treatment. [ABSTRACT FROM AUTHOR]

The article focuses on the potential use of quercetin, a flavonoid, as a senolytic molecule to counteract cellular senescence, particularly in human mesenchymal stromal/stem cells (MSCs), by activating autophagy and apoptosis in senescent cells.

The article focuses on the pro-differentiating effects of red photobiomodulation on skeletal myoblasts, with evidence suggesting its potential to enhance myoblast differentiation while maintaining myotube viability and size.

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes the in vitro behavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2 ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes. [ABSTRACT FROM AUTHOR]

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes the in vitro behavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2 ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes. [ABSTRACT FROM AUTHOR]

Bone tissue engineering approaches are encouraging for the improvement of conventional bone grafting technique drawbacks. Thanks to their self-renewal and multi-lineage differentiation ability, stem cells are one of the major actors in tissue engineering approaches, and among these adult mesenchymal stem cells (MSCs) hold a great promise for regenerative medicine strategies. Bone marrow MSCs (BM-MSCs) are the first- identified and well-recognized stem cell population used in bone tissue engineering. Nevertheless, several factors hamper BM-MSC clinical application and subsequently, new stem cell sources have been investigated for these purposes. The fruitful selection and combination of tissue engineered scaffold, progenitor cells, and physiologic signaling molecules allowed the surgeon to reconstruct the missing natural tissue. On the basis of these considerations, we analyzed the capability of two different scaffolds, planned for osteochondral tissue regeneration, to modulate differentiation of adult stem cells of dissimilar local sources (i.e., periodontal ligament, maxillary periosteum) as well as adipose-derived stem cells (ASCs), in view of possible craniofacial tissue engineering strategies. We demonstrated that cells are differently committed toward the osteoblastic phenotype and therefore, taking into account their specific features, they could be intriguing cell sources in different stem cell-based bone/periodontal tissue regeneration approaches. [ABSTRACT FROM AUTHOR]

ABSTRACT Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system. Microsc. Res. Tech. 77:928-934, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background The role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance, both for the enhanced understanding of fundamental cancer biology as well as for improving molecular diagnostics and therapeutics. The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells. Methods The HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24 h to 7 days. Cell morphology and organization were studied using phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloprotease 1 (MMP1) was investigated by Real Time PCR and Western Blotting. Results The results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured. Conclusions We concluded that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity. [ABSTRACT FROM AUTHOR]

Novel polylactide (PLA) microspheres endowed with hydrophilic and bioadhesive surfaces as injectable formulations for the controlled release of fenretinide were prepared, using a novel technique based on the co-precipitation of PLA with gelatin, at the interface of a liquid dispersion formed by the addition of N-methylpyrrolidone containing PLA and dextrin (DX), towards an aqueous solution of gelatin (G). The resulting PLA-G-DX microspheres were compared with others prepared by the same technique using polylactide-co-glycolide (PLGA), with or without DX, and with or without phosphatidylcholine. Of the different systems, the PLA-G-DX microspheres had the best morphological, dimensional and functional characteristics. They had the highest drug loading, and their drug release was the most efficient over time without any burst effect. Their in vitro anti-tumoural activity was strongly enhanced with respect to the pure fenretinide. This paralleled the increased drug concentration inside the cells due to their marked bioadhesion to the tumour cell membranes as indicated by scanning electron microscope images. [ABSTRACT FROM AUTHOR]

The main purpose of this article was to describe the morphology of mesenchymal stem cells (MSCs) differentiated in vitro towards osteogenic and chondrogenic lineages and to focus on the ultrastructural features associated with these processes. Human mononuclear cells (hMNC) were isolated, expanded, and analyzed for the expression of specific cell surface markers to demonstrate their stem cell characteristics. Human mononuclear cells were differentiated in vitro in an osteogenic and in a chondrogenic sense for 7, 14, 21, and 28 days. Subsequently, they were processed using electron microscopic analysis (FEISEM). Alizarin red and alcian blue staining were carried out to demonstrate the deposition of mineral salts and proteoglycans in the extracellular matrix. Undifferentiated MSCs showed a cell surface covered by filopodia and ondulopodia. During differentiation, the MSCs changed their shape from a round to a fibroblastic-like shape. At the end of the differentiation, several filaments with a parallel orientation in the osteogenic samples as well as a network organization in the chondrogenic samples were detected in the extracellular spaces. This study demonstrated that there are morphological features associated with the undifferentiated and differentiated states of the MSCs, which could be utilized as new parameters for identifying and classifying these cells. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the cytotoxicity of low doses and long-term exposure to 2-hydroxyethylmethacrylate (HEMA) on the protein expression of human gingival fibroblasts (HGFs). Human gingival fibroblasts were exposed to different concentrations of HEMA ranging from 0.5 mmol/L to 3 mmol/L for periods of time from 72 hours to 2 weeks. A significant decrease in the expression of procollagen α1 type I protein was observed 72 hours after treatment of cells with 3 mmol/L HEMA. Although low concentrations of the monomer after 2 weeks of exposure to HEMA did not appear to induce any marked changes in the morphology or viability of cells, the expression of procollagen α1 type I protein and its messenger RNA (mRNA) markedly decreased. In conclusion, our data demonstrated that cell viability and morphology assays could be deficient parameters in evaluating the biocompatibility of dental resin materials. [ABSTRACT FROM PUBLISHER]

Venerina (little Venus) is the name given to a wax model representing a pregnant young woman that was created in Florence (Italy) by Clemente Susini (1754–1814) in 1782. It is currently located in the historic Science Museum of the University of Bologna. The model was constructed so as to enable removal of the thoracic and abdominal walls and various organs, exposing the heart, diaphragm and an opened uterus with a well-developed fetus. The woman is small, about 145 cm (4′ 9′) tall and of delicate build; she looks like a teenage girl. We know that Clemente Susini worked directly with the cadaver and copied the anatomical preparation exactly. This artist often represented the true structure using a wax mould; the existence of two other versions of this specimen suggests that this model was made in this way. Therefore, Venerina’s body may be a faithful representation of a young woman who died while pregnant. Observation of the body confirms that the organs are normal, except for the heart and great vessels. The walls of both ventricles are of equal thickness and the ventricles themselves of approximately equal size. The arch of the aorta and the enlarged pulmonary trunk are connected by a short duct about 3.5 mm in diameter. If this structure represents an open arterial duct, we can deduce that the two ventricles worked under the same conditions of blood pressure, hence their equal wall thickness. If the young woman died from this congenital disease, the cause of death has been diagnosed on a wax model of her body after more than two centuries. [ABSTRACT FROM AUTHOR]

Sulforaphane (SF), one of the most important isothiocyanates in the human diet, present in cruciferous vegetables, is known to have chemopreventive activities in different tissues. No data are available on its effects in the prevention of skeletal muscle damage. In this study, we investigated the potential protective effects of SF treatment on muscle damage and oxidative stress induced by an acute bout of exhaustive exercise in rats. Male Wistar rats were treated with SF (25 mg/kg body wt ip) for 3 days before undergoing an acute exhaustive exercise protocol in a treadmill (+7% slope and 24 m/min). Acute exercise resulted in a significant increase in plasma lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activities. It also resulted in a significant increase in thiobarbituric acid-reactive substances, in a significant decrease in tissue total antioxidant capacity, and in a significant decrease in NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in vastus lateralis muscle. SF treatment significantly increased muscle NQO1, glutathione-S-transferase, and glutathione reductase expression and activity, with no effect on glutathione peroxidase and thioredoxin reductase. The observed SF induced upregulation of phase II enzymes was accompanied by a significant increase in nuclear erythroid 2 p45-related factor 2 expression and correlated with a significant increase in total antioxidant capacity and a decrease in plasma LDH and CPK activities. Our data demonstrate that SF acts as an indirect antioxidant in skeletal muscle and could play a critical role in the modulation of the muscle redox environment, leading to the prevention of exhaustive exercise-induced muscle damage. [ABSTRACT FROM AUTHOR]

Tattooing is an ancient art and is still widely practiced all over the world. Since the biocompatibility of tattoo dyes has not been well researched, we studied the toxicity of a commercial tattoo ink, commonly used in tattoo lab and esthetic centers, on human fibroblasts. To test cell viability, MTT assays were carried out and scanning electron microscopy to visualize changes in the cell surface after the dye exposure was performed. A possible influence of the pigment on the expression of procollagen α1 type I protein was visualized by western blotting analysis. The results showed a reduction in cell viability, and electron microscopy demonstrated an unmodified cell surface completely covered by pigment particles. Western blotting analysis demonstrated a clear interference of the pigment on the expression of procollagen α1 type I protein. These data demonstrated that the commercial tattoo dye has a time-dependent effect on protein expression. A possible connection of the influence of the tattoo ink with clinical effects is discussed. [ABSTRACT FROM AUTHOR]

Inositide-specific phospholipase Cβ1 PLCβ1) signaling in cell proliferation has been investigated thoroughly in the G1 cell cycle phase. However, little is known about its involvement in G2/M progression. We used murine erythroleukemia cells to investigate the role of PLCβ1 in G2/M cell cycle progression and screened a number of candidate intermediate players, particularly mitogen-activated protein kinase (MAPK) and protein kinase C (PKC), which can, potentially, transduce serum mitogenic stimulus and induce lamin B1 phosphorylation, leading to G2/M progression. We report that PLCβ1 colocalizes and physically interacts with lamin B1. Studies of the effects of inhibitors and selective si-RNA mediated silencing showed a role of JNK, PKCα, PKCβ1, and the β1 isoform of PI-PLC in cell accumulation in G2/M [as observed by fluorescence-activated cell sorter (FACS)]. To shed light on the mechanism, we considered that the final signaling target was lamin B1 phosphorylation. When JNK, PKCα, or PLCβ1 were silenced, lamin B1 exhibited a lower extent of phosphorylation, as compared to control. The salient features to emerge from these studies are a common pathway in which JNK is likely to represent a link between mitogenic stimulus and activation of PLCβ1, and, foremost, the finding that the PLCβ1-mediated pathway represents a functional nuclear inositide signaling in the G2/M transition. [ABSTRACT FROM AUTHOR]

In this study, we analyzed the chromatin ultrastructure in interphase cells after different chemical fixations. In light of the fact that there is little information regarding the fixation of biological samples in combination with molecular biology methods (such as DNA extraction and in situ hybridization methods) we analyzed the ultrastructure of chromatin in interphase cells fixed with different fixatives and tested under the same conditions for both DNA extraction and in situ hybridization. The results showed that, among the different combinations and concentrations we analyzed, the solution of 4% paraformaldehyde/0.1% glutaraldehyde was the best compromise in order to achieve a well-preserved morphology, successful DNA extraction, and specific signaling of in situ hybridization, suggesting a low interference of this fixative with the chromatin organization. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The article presents a study on equine synovial fluid mesenchymal stromal cells. Topics include information on implication of cellular senescence in osteoarthritis (OA); information on most common degenerative orthopedic disease in humans; and how cell therapy based on the application of mesenchymal stem/stromal cells (MSCs) and/or their secretome represents tool for OA treatment.

The article offers information on potential markers of skin wound vitality in forensic practice. Topics include information on NFkB and SIRT1 evaluation to discriminate antemortem lesions from postmortem damages in wounds; causal relationship between the cause of death and wounds; and how quantitative analysis of stained areas is done.

Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene. This mutation leads to the production of mutant HTT (mHTT) protein which triggers neuronal death through several mechanisms. Here, we investigated the neuroprotective effects of esculetin (ESC), a bioactive phenolic compound, in an inducible PC12 model and a transgenic Drosophila melanogaster model of HD, both of which express mHTT fragments. ESC partially inhibited the progression of mHTT aggregation and reduced neuronal death through its ability to counteract the oxidative stress and mitochondria impairment elicited by mHTT in the PC12 model. The ability of ESC to counteract neuronal death was also confirmed in the transgenic Drosophila model. Although ESC did not modify the lifespan of the transgenic Drosophila, it still seemed to have a positive impact on the HD phenotype of this model. Based on our findings, ESC may be further studied as a potential neuroprotective agent in a rodent transgenic model of HD. [ABSTRACT FROM AUTHOR]

The discovery of opioid receptors expression in skin and their role in orchestrating the tissue repairing process, gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve wounds regeneration, in vivo chronic administration was associated to immune cell recruitment delay and abnormal extracellular matrix deposition, impairing the physiological tissue healing progression and causing systemic fibrosis. Human mesenchymal stem cells (hMSCs) are a versatile class of multipotent adult stem cells. Their self-renew and multidirectional differentiation ability, combined with the release of immunoregulatory molecules, make them fundamental in restoring the damaged tissues. In this regard, opioid receptors expression was recently observed even in hMSCs[1], for which acute morphine exposition induced a significant functional characteristics decline[2]. However still little is known about its long-term effects. To determine how a prolonged treatment could impair their regenerative potential, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential. Our results showed a time dependent cell viability decline and conditions compatible with a cellular senescent state. Ultrastructural analysis combined with beclin-1 (BECN1) and microtubuleassociated proteins 1A/1B light chain 3B (LC3) expression, after eighteen days of exposition, were indicative of increased autophagy, suggesting a correlation to an augmented detoxification activity. In addition, the enhanced transcription of type I collagen (COL1A1) together with the related genes involved in its regulation, namely: metalloproteinase-1 and -2 (MMP1, MMP2), transforming growth factor-beta1 (TGFB1) and tumour necrosis factor-alpha (TNFA), suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk even involving the hMSCs. [ABSTRACT FROM AUTHOR]

Abdominal aortic aneurysm (AAA) is an age-related disease characterized by chronic inflammation, increased local activation of matrix-degrading proteinases, and weakening of the vascular wall. The main complication is the rupture of the abdominal aortic wall which leads to death. Nowadays, no pharmacological therapies are available and the understanding of the molecular mechanisms that lead to AAA onset and its development are poorly defined. Reparative abilities of vascular mesenchymal stromal cells (MSCs) have a key role in vascular remodeling. However, unbalanced activity of vascular MSCs support AAA pathogenesis. Cellular senescence is considered one of the main hallmarks of aging. It is classified as an irreversible growth arrest of the cell cycle which has beneficial roles in physiological conditions. However, the accumulation of senescent cells during aging has adverse consequences and it is believed to have a key role in the onset and development of several age-related diseases. The aim of this study was to demonstrate the presence of cellular senescence in vascular MSCs isolated from AAA segments (AAA - MSCs), responsible of an impaired vascular remodeling process. AAA-MSCs were assayed for their proliferative ability, cellular senescence markers, autophagy and in vitro vascular differentiation. The results were compared to MSCs isolated from healthy segment of the abdominal aorta of the same patients (h - MSCs). The results from AAA-MSCs clearly demonstrated a reduced proliferation ability, an increase of ROS levels, the positive expression of the senescent markers p21CIP1 and p16INK4a, a dysregulated autophagy and a strongly impaired ability in differentiating toward an endothelial phenotype. All these results indicate the presence of senescent vascular MSCs in the wall of AAA and strongly support the hypothesis that an accumulation of senescent vascular MSCs could have a pivotal role in the onset and development of AAA. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result, or possibly originate, from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore, we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether, our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses. [ABSTRACT FROM AUTHOR]

Osteoarthritis is a degenerative disease that strongly correlates with age and promotes the breakdown of joint cartilage and subchondral bone. There has been a surge of interest in developing cell-based therapies, focused particularly on the use of mesenchymal stromal cells (MSCs) isolated from adult tissues. It seems that MSCs derived from synovial joint tissues exhibit superior chondrogenic ability, but their unclear distribution and low frequency actually limit their clinical application. To date, the influence of aging on synovial joint derived MSCs' biological characteristics and differentiation abilities remains unknown, and a full understanding of the mechanisms involved in cellular aging is lacking. The aim of this study was therefore to investigate the presence of age-related alterations in synovial fluid MSCs and their influence on the potential ability of MSCs to differentiate toward chondrogenic phenotypes. Synovial fluid MSCs, isolated from healthy equine donors from 3 to 40 years old, were cultured in vitro and stimulated towards chondrogenic differentiation for up to 21 days. An equine model was chosen due to the high degree of similarity of the anatomy of the knee joint to the human knee joint and as spontaneous disorders develop that are clinically relevant to similar human disorders. The results showed a reduction in cell proliferation correlated with age and the presence of age-related tetraploid cells. Ultrastructural analysis demonstrated the presence of morphological features correlated with aging such as endoplasmic reticulum stress, autophagy, and mitophagy. Alcian blue assay and real-time PCR data showed a reduction of efficiency in the chondrogenic differentiation of aged synovial fluid MSCs compared to young MSCs. All these data highlighted the influence of aging on MSCs' characteristics and ability to differentiate towards chondrogenic differentiation and emphasize the importance of considering age-related alterations of MSCs in clinical applications. [ABSTRACT FROM AUTHOR]

Abdominal aortic aneurysm (AAA) is a common degenerative vascular disorder associated with sudden death due to aortic rupture. The current clinical approaches are to monitor aortic dimensions and to perform an open or endovascular surgical repair when the aortic diameter has attained sufficient expansion, condition that predispose to a high likelihood of aortic rupture. Although several studies have identified many potential mechanisms involved in AAA pathogenesis, a clear depth understanding is still lacking and further studies are needed to facilitate development of effective therapies. Recent discoveries have demonstrated the presence of mesenchymal stromal cells (MSCs) in human aortic layers. These cells possess high proliferative capacity and potential to generate endothelial, smooth muscle, hematopoietic and mesenchymal cell progeny. Nevertheless, any defect of the proliferation and/or the differentiation process of vascular stem cells may determine the development of human vascular diseases. The aim of this study was to demonstrate the presence of senescent MSCs residing in human abdominal aortic wall, which could have a role in the AAA pathogenesis. MSCs isolated from healthy (HAA - MSCs) or aneurysmal abdominal aortas (AAA - MSCs) were characterized for their proliferation rate, ultrastructural morphology, senescence-associated β-galactosidase activity and differentiation properties. Results showed low growth potential, high senescence-associated β-galactosidase activity, an increased cell surface area, a reduced amount of autophagic and lysosome vesicles in AAA - MSCs compared to HAA - MSCs, thus indicated a senescent phenotype in AAA MSCs. Vascular wall-resident MSCs are deeply involved in the process of vascular remodelling, that is a dynamic and strictly regulated process of structural changes occurs as a result of a pathological vascular trigger. The presence of a senescent population of AAA MSCs in vascular wall could have implications in the genesis and progression of vascular diseases, such as AAA. [ABSTRACT FROM AUTHOR]

Human dentin is formed during the process of dentinogenesis synthesized by odontoblasts that, in addition to type I collagen, also secrete a number of non-collagenous proteins (NCPs) into extracellular matrix (ECM) during the process of dentinogenesis. The Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family is one category of NCPs characteristic for dentin and bone including dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), and osteopontin (OPN) [1]. Tertiary dentin is produced in reaction to external noxious stimulus/injury, such as trauma ordental caries and based on the type and extent of external stimuli or injuries, is further classified into reactionary dentin (RD) and reparative dentin (RepD). Aim this study was to compare pattern distribution and quantification of SIBILINGs in reparative and reactionary dentin matrix, in response to stimulus, vs human sound dentin. Ten carious human molars and ten sound human molars were selected for the study, demineralized, fixed, and processed for immunohistochemical approach to detect SIBILINGs. In particular specimens were submitted to an immunolabeling technique by using primary antibodies anti dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), osteoponti (OPN), observed at light microscopy and then submitted to quantitative analysis. Results indicate that the region exposed to carious lesion, SIBILINGs formed a layer of reparative dentin bridge sealing cavity formed by carious lesion and pulp chamber. In response to the injury, the newly differentiated odontoblast-like cells makes adjustments to meet the challenges by altering the production of these dentinogenesis-related molecules, attempting to produce a hard barrier formation in a very rapid process. [ABSTRACT FROM AUTHOR]

Articular cartilage is an avascular tissue without innervations, characterized by low cell density and abundant extracellular matrix (ECM). These characteristics leave articular cartilage with very limited capacity of repair and regeneration. Multipotent stem/stromal cells (MSC) are considered promising for cartilage tissue engineering. Stem cells are resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Previous studies have reported that hypoxic conditions could enhance the chondrogenic differentiation of mesenchymal stem cells in the presence of an inductive medium. Cobalt chloride (CoCl2) imitates hypoxia in vitro by preventing hypoxiainducible factor-alpha (HIF-a) from being destroyed by oxygen. However, the longterm hypoxic culture of stem cells is difficult and requires special attention to avoid cell death due to cobalt treatment. In this study we investigated if CoCl2 affected MSCs isolated from dental pulp, umbilical cord and adipose tissue in their potential to differentiate toward the chondrogenic phenotype. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 uM. Cell proliferation, mRNA expression of stem-cell marker and chondrogenic associated genes were analyzed by RT-PCR and Real-time PCR. The results showed that the CoCl2 supplementation had no effect on the proliferation of all the three type of cells analyzed, while the up-regulation of chondrogenic markers such as aggrecan, sox9, and type II collagen, was dependent on the cellular source. This study shows that hypoxia induced by CoCl2 treatment can differently influence the behavior of MSCs of different sources in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies. [ABSTRACT FROM AUTHOR]

The estimation of the post-mortem interval (PMI) is a critical step in forensic medicine and remains one of the most challenging variables to determine. In general, the numerous methods currently used in estimating post-mortem interval yield to large post-mortem windows, are influenced by several factors and sometime contradict each other. With the aim to obtain a much more accurate determination of the post-mortem interval we combined morphological ultrastructural and immunoistochemical analyses to reach a more detailed knowledge on tissue organization and degradation after death. Samples of gingival tissues obtained from 20 cadavers at different post-mortem intervals were processed for transmission electron microscopy to evaluate ultrastructural modifications in the epithelium and connective tissue. Gingival cells and the extracellular matrix of gingival tissues have been observed by a transmission electron microscopy (TEM) in combination with the evaluation of potential post-mortem biochemical markers, with the final goal to find a tight correlation between the ultrastructural modifications, the biomarker expression and the time of death. All the samples were also immunostained with anti-hypoxia-induced factor 1-α (HIF1-α) antibody, a transcription factor expressed in response to hypoxia, in order to evaluate the expression of HIF1-α, and to establish a correlation between the protein presence and the time of death. Results showed nuclear chromatin changes and cytoplasmic vacuolization in both epithelial and connective tissues and a different pattern of expression of HIF1alpha protein that correlate to the time of death. In conclusion, our preliminary findings suggest that ultrastructural investigations in combination with immunohistochemistry techniques in gingival specimens may represent a new tool to accurately and systematically estimate post-mortem interval. [ABSTRACT FROM AUTHOR]