68 research outputs found

    Effect of aging on extracellular matrix and collagen turnover related pathways in human tendons and cultured human tenocytes

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    The aging process involves different organs, including the musculoskeletal system. Age-related modifications affecting the tendon such as reduced cell proliferation and decreased glycosaminoglycan and proteoglycan content were previously described, but data are incomplete and discordant. Therefore, aim of our study was to characterize the effect of aging on tendons, with particular attention to collagen turnover. For this purpose, tendons and cultured tenocytes were obtained by healthy young (age <65 years) and aging subjects (≥65 years), and analyzed by morphological and molecular methods. Our data show that aging tenocytes have a reduced proliferation rate. Haematoxylin and eosin, Sirius red and Alcian blue staining, respectively, revealed that tendon structure is maintained, and that collagen and proteoglycan content is similar in young and aging tendons. However, decreased lysyl hydroxylase 2b gene expression was observed in aging tenocytes, suggesting that differences in collagen maturation could be responsible for a decreased ability to resist mechanical loading. By fluorescence microscopy, actin cytoskeleton modifications such as fewer and shorter stress fibers were observed in some aging tenocytes, consistent with a decreased ability to form focal adhesions and, therefore, a reduced migration potential. Intermediate filaments and microtubules were not modified by aging. Considered as a whole, our results suggest that the structure of aged tendons is preserved, but the biomechanical properties could be impaired by reduced collagen cross-linking. Moreover, modifications of actin filaments could affect the mechanotransduction system allowing tenocytes to adapt their ability for extracellular matrix remodeling in response to mechanical loading. Therefore, aged tendons could be likely more prone to develop tendinopathies

    Polyblend Nanofibers to Regenerate Gingival Tissue: A Preliminary In Vitro Study

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    Aim: The regeneration of small periodontal defects has been considered an important divide and challenging issue for dental practitioners. The aim of this preliminary in vitro study was to analyze the effects of polycaprolactone (PCL) nanofibers enriched with hyaluronic acid and vitamin E vs. nude nanofibers on gingival fibroblasts activity, an innovative graft for periodontal soft tissue regeneration purposes. Methods: Nanofibers were produced in PCL (NF) or PCL enriched with hyaluronic acid and vitamin E (NFE) by electrospinning technique. NF and NFE were stereologically and morphologically characterized by scanning electron microscope (SEM), and composition was analyzed by infrared spectroscopy. Human fibroblasts were obtained from one gingival tissue fragment (HGF) and then seeded on NF, NFE, and plastic (CT). Cell adhesion and morphology were evaluated using SEM at 24 h and cell viability after 24, 48, and 72 h by alamarBlue® assay. Gene expression for COL-I, LH2b, TIMP-1, PAX, and VNC was analyzed by real-time RT-PCR in samples run in triplicate and GAPDH was used as housekeeping gene. Slot blot analysis was performed and immunoreactive bands were revealed for MMP-1 and COL-I. YAP and p-YAP were analyzed by Western blot and membranes were reprobed by α-tubulin. Statistical analysis was performed. Results: IR spectrum revealed the presence of PCL in NF and PCL and vitamin E and hyaluronic acid in NFE. At 24 h, HGF adhered on NF and NFE conserving fibroblast like morphology. At 72 h from seeding, statistically significant differences were found in proliferation of HGF cultured on NF compared to NFE. Expression of genes (LH2b, TIMP-1, and MMP-1) and proteins (COL-I) related to collagen turnover revealed a reduction of COL-1 secretion in cells cultured on NF and NFE compared to CT; however, NFE stimulated cross-linked collagen deposition. Mechanosensor genes (PAX, VNC, and YAP) were upregulated in HGF on NF while they were decreased in cells grown on NFE. Conclusion: Preliminary data suggest that PCL-enriched nanofibers could represent a support to induce HGF proliferation, adhesion, collagen cross-linking, and to reduce collagen degradation, therefore favoring collagen deposition in gingival connective tissue

    Clinical, histological, immunohistochemical and biomolecular analysis of hyaluronic acid in early wound healing of human gingival tissues: a randomized, split‐mouth trial

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    Background: Hyaluronic acid (HA) exerts a fundamental role in tissue repair. In vitro and animal studies demonstrated its ability to enhance wound healing. Nevertheless, in vivo human studies evaluating mechanisms involved in oral soft tissue repair are lacking. The aim of this study was to evaluate the in vivo effect of HA on early wound healing of human gingival tissues (G). Methods: In the present randomized, split-mouth, double-blind, clinical trial, G biopsies were obtained in eight patients 24 hours after surgery after HA application (treatment group-HA) and compared with those obtained from the same patients without HA application (no treatment group-NT). Clinical response was evaluated through Early Wound Healing Score (EHS). Microvascular density (MVD), collagen content and cellular proliferation were evaluated through Sirius red, Masson trichrome staining and Ki67 immunohistochemistry, respectively. To assess collagen turnover, MMP-1, MMP-2, MMP-9, TGF-β1 protein levels and LOX, MMP-1, TIMP-1, TGF-β1 gene expression were analysed by Western Blot and Real Time PCR. Results: Twenty-four hours after surgery, EHS was significantly higher in HA group. MVD, collagen content and cell proliferation were not affected. LOX mRNA, MMP-1 protein and TIMP-1 gene expression were significantly up-regulated in HA compared to NT group. Conclusions: The additional use of 0.8% HA gel does not modify the new blood vessels growth in the early phase of gingival wound healing. Concerning the secondary outcomes, HA seems to enhance extracellular matrix remodelling and collagen maturation, that could drive the early wound healing of gingival tissues to improve clinical parameters. This article is protected by copyright. All rights reserved

    Effect of extracellular matrix components on the expression of epithelial-to-mesenchymal transition markers in cultured human pancreatic ductal adenocarcinoma cells

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    Epithelial-to-mesenchymal transition (EMT) is a step-wise process leading to the phenotypic switch of epithelial to mesenchymal cells, providing these cells with a metastatic phenotype. During EMT epithelial cells loose adhesion by down regulation of E-cadherin and express N-cadherin, display cytoskeleton reorganization by expressing vimentin and α-smooth muscle actin (αSMA), acquire motile properties and become invasive by secretion of matrix metalloproteinases (MMPs). Cancer cell phenotype is influenced by the tumor microenvironment in relation to tumor progression, as well as to cell proliferation and invasion. The role of the extracellular matrix (ECM) in the microenvironment is particularly relevant in pancreatic ductal adenocarcinoma (PDAC) since this carcinoma is characterized by an intense desmoplastic reaction, representing the environment where the complex interplay between tumor cells, stromal fibroblasts and ECM components occurs. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and their microenvironment by characterizing PDAC cell phenotype in cells cultured on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell behaviour and the proteins occurring in the desmoplastic tissue. We analyzed by immunofluorescence the expression of the main EMT markers such as E-cadherin, N-cadherin, β-catenin, αSMA, vimentin and collagen type I (COL-I) in PDAC cells cultured on laminin, fibronectin, COL-I and without coating (NC). Moreover, we investigated cell proliferation and MMPs activity in cell culture supernatants by SDS-zymography. Cell morphology was similar in PDAC cells cultured on laminin, fibronectin, COL-I, and in NC, as well as the E-cadherin/β-catenin complex, αSMA and COLI expression; by contrast, vimentin was undetectable in all the experimental conditions. N-cadherin was slightly detectable in cells cultured on fibronectin, COL-I, and laminin, and at lower extent in NC cells. Cell proliferation resulted similar in NC and in cells cultured on fibronectin, decreased on laminin and increased on COL-I. MMP-9 activity exhibited a similar trend, resulting similar on fibronectin, decreased on laminin and stimulated on COL-I. These preliminary results provide new insights in the characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell, and will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies

    Skin morphological analysis upon treatment with anti-TNF-alpha agents in psoriatic patients

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    Psoriasis is the most common immune-mediated skin disease worldwide, with an estimated incidence of about 2%. It is characterized by erythematous scaly plaques and its pathogenesis is mostly due to an interplay among epidermal cells, immunocompetent cells, and pro-inflammatory cytokines. Previous studies on psoriatic skin demonstrated delayed terminal differentiation, keratinocyte hyperproliferation, and abnormal occludin expression in tight junctions. However, evidences about desmosomal cadherin distribution in psoriatic patients are not available so far, and only scattered studies have been carried out on adherens junctions. In psoriasis, Tumor Necrosis Factor- alpha (TNF-alpha) plays a central role, strongly supporting the treatment with anti-TNF-alpha agents, but the effects of these agents on epidermal intercellular adhesion, terminal differentiation, and proliferation have still to be elucidated. In this preliminary study, we investigated by immunofluorescence the expression of transmembrane proteins in tight junctions (occludin), adherens junctions (E-cadherin), and desmosomes (desmocollin-1 and desmoglein-1) in normal (N=5) and psoriatic skin before/after treatment with anti-TNF-alpha agents (N=5). Differentiation biomarkers (keratin-10, keratin-14, and involucrin) and epithelial proliferation were also evaluated. In psoriatic epidermis occludin, keratin-14, and involucrin were expressed also in the spinous layer, differently from controls. Desmoglein-1, desmocollin-1, E-cadherin, and keratin-10 localizations were comparable in psoriatic and healthy subjects. Moreover, in all considered patients the hyperproliferative condition was accompanied by an unusual suprabasal distribution of replicating keratinocytes. Interestingly, the distribution pattern of all considered biomarkers of intercellular adhesion and terminal differentiation was reverted to the physiological condition upon treatment with anti-TNF-alpha agents. What’s more, the proliferative rate registered in anti-TNF-alpha treated patients was reduced and similar to controls, even though scattered suprabasal dividing cells were still present. Our results highlight that anti-TNF-alpha biologicals, next to their proven inhibitory action on the cytokinic pathway, are effective in restoring an efficient junctional apparatus, a more differentiated phenotype, and the typical proliferation rate in the epidermis of psoriatic patients

    Extracellular matrix components affect cell migration and invasive potential of cultured human pancreatic ductal adenocarcinoma cells

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    The tumor microenvironment influences cancer cell behavior in relation to tumor progression, as well as cell proliferation and invasion. Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense desmoplastic reaction and extracellular matrix (ECM) components in the tumor microenvironment are involved in a cross-talk between tumor cells, stromal fibroblasts and ECM components, influencing tumor cell behavior. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and the ECM of the microenvironment by culturing PDAC cells on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell phenotype and the proteins occurring in the desmoplastic tissue. For this purpose, we analyzed some epithelial-to-mesenchymal transition (EMT) markers and the migration and invasive potential in human HPAF-II, HPAC and PL45 PDAC cells cultured on collagen type I (COL), laminin (LAM) and fibronectin (FN). Interestingly, the expression of E-cadherin was not significantly affected, but some differences were revealed by the wound healing assay. In fact, migration of HPAF-II and PL45 cells was decreased on FN and LAM, and increased on COL, compared to control cells grown on plastic (NC). By contrast, HPAC was very rapid and unaffected by the substrate. SDS-zymography showed that COL induced a strong upregulation of MMP-2 activity in HPAF-II and HPAC cells, and of MMP-9 in HPAF-II and PL45 cells, compared to NC. These preliminary results suggest that ECM components could differently affect PDAC migration and invasion, possibly depending on the differentiation grade. The characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies

    Analysis of tissue structure and remodeling ion alveolar ridges augmented with human palate or tuberosity mucosa

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    Previous clinical reports found different clinical outcomes of localized alveolar ridge augmentation with soft tissue harvested either from the palate or from the tuberosity over time, showing that the palatal grafts had a better tissue stability than those from the tuberosity, which tended to a hyperplastic reaction. The mechanisms responsible for a different maturation of the grafted tissue using the two donor sites are still unclear, very likely depending on differences of the structure and extracellular matrix of connective tissue (CT). The current study aimed at comparing the morphology and collagen turnover-related molecular pathways of sites grafted with CT from either the palate (group A = 7 patients) or the tuberosity (group B = 7 patients) one year after surgical procedures for ridge augmentation. Cultured fibroblasts were obtained to analyze by real-time PCR the mRNA levels for collagen type I and III (COL-I, COL-III), matrix metalloproteinases (MMP-1 and 2), long lysyl hydroxylase 2b (LH2b). Collagen protein levels were assessed by slot blot, collagen degradation by SDS-zymography. No significant differences in collagen content were found. COLI and III, MMP-1 and 2 expression was similar in cell culture supernatants from palate and tuberosity fibroblasts, although COL-I and COL-III protein levels resulted up-regulated, respectively, in 57% and 66% of the samples. LH2b/COL-I mRNA ratio 69% was higher in the tuberosity fibroblasts, suggesting that the tuberosity collagen could be cross-linked at a higher extent, and therefore less susceptible to degradation by MMPs, leading to its excessive accumulation. Our data show that in group B the higher LH2b/COL-I mRNA ratio may be responsible for differences in collagen maturation as the major determinants in the hyperplastic response, and that grafting using the maxillary tuberosity could avoid unwanted tissue contraction over time

    Characterization of an in vitro model to study the role of human Polyomavirus BK in prostate cancer

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    Prostate cancer (PCa) is one of the most common male neoplasm in the western world, being the most commonly diagnosed non-skin cancer and the second leading cause of cancer death. Various potential risk factors exist for the initial triggering events, including exposure to infectious agents, such as the human Polyomavirus BK (BKV). BKV is a good candidate as risk factor of PCa because it naturally infects the human reno-urinary tract, it establishes latency, and encodes oncoproteins that interfere with tumor suppressors pathways, thus altering the normal progression of cell cycle. Previous studies suggested a potential association between BKV and PCa, revealing that the prevalence of BKV was significantly higher in cancer than in control tissues, with a significant association between viral expression and cancer. However, this hypothesis is controversial because BKV is not restricted to tumor tissues but also infects healthy individuals in a high percentage. Moreover, an in vitro model of BKV infection in prostate cells is not available to understand the role for BKV in pathogenesis of PCa. Our aims were to determine whether BKV a) could infect normal epithelial prostate cells, b) affects cell phenotype and c) affects the phenotype of human prostate tumor cell line PC3. For this purpose normal epithelial prostate cell line RWPE-1 and prostate cancer cells PC3 were infected with BKV for 21 days. Cell proliferation, epithelial-to-mesenchymal markers (EMT) and invasion potential were analyzed by, respectively, MTT, immunofluorescence and SDS-zymography. Our results show that cell proliferation was increased or decreased by BKV, respectively, in RWPE-1 and PC3 cells. BKV induced E-cadherin downregulation and vimentin expression in both control and BKV-infected cells RWPE-1, suggesting that uninfected cells underwent EMT. Matrix metalloproteinase-2 and 9 activity was increased in RWPE-1 cells after BKV infection. By contrast, BKV did not significantly modified the phenotype of PC3 cells. These preliminary results suggest that normal epithelial prostate cells RWPE-1 and PC3 are susceptible and permissive to BKV infection. However, RWPE-1 cells exhibit some phenotype modifications related to EMT, possibly induced by the papilloma virus used to obtain their immortalization, thus suggesting that further experiments will be necessary to define if they represent a good experimental model to study prostate cancer
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