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Mechanical force promotes the proliferation and extracellular matrix synthesis of human gingival fibroblasts cultured on 3D PLGA scaffolds via TGF‑β expression.

Human gingival fibroblasts (HGFs) are responsible for connective tissue repair and scarring, and are exposed to mechanical forces under physiological and pathological conditions. The exact mechanisms underlying gingival tissue reconstruction under mechanical forces remain unclear. The present study aimfed to investigate the effects of mechanical forces on the proliferation and extracellular matrix synthesis in HGFs by establishing a 3‑dimensional (3D) HGF culture model using poly(lactide‑co‑glycolide) (PLGA) scaffolds. HGFs were cultured in 3D PLGA scaffolds and a mechanical force of 0, 5, 15, 25 or 35 g/cm2 was applied to HGFs for 24 h. A mechanical force of 25 g/cm2 induced the highest proliferation rate, and thus was selected for subsequent experiments. Cell viability was determined using the MTT assay at 0, 24, 48 and 72 h. The expression levels of type I collagen (COL‑1) and matrix metallopeptidase (MMP)‑1 were examined by reverse transcription‑quantitative polymerase chain reaction and ELISA, and transforming growth factor (TGF)‑β expression was evaluated by ELISA. The application of mechanical force on HGFs cultured on the 3D PLGA scaffolds resulted in a significant increase in cell proliferation and COL‑1 expression, as well as a decrease in MMP‑1 expression. A TGF‑β1 inhibitor was also applied, which attenuated the effects of mechanical force on HGF proliferation, and COL‑1 and MMP‑1 expression, thus suggesting that TGF‑β signaling pathways may mediate the mechanical force‑induced alterations observed in HGFs. In conclusion, these findings helped to clarify the mechanisms underlying mechanical force‑induced HGF proliferation and ECM synthesis, which may promote the development of targeted therapeutics to treat various diseases, including gingival atrophy caused by orthodontic treatment.

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Photobiomodulation in the Metabolism of Lipopolysaccharides-exposed Epithelial Cells and Gingival Fibroblasts.

This study assessed the effects of photobiomodulation (PBM) to cells previously exposed to lipopolysaccharides (LPS). Human gingival fibroblasts (HGF) and epithelial cells (HaCaT) were seeded in wells of 24-well plates containing complete culture medium (DMEM). After 24 h, the DMEM was replaced by serum-free DMEM, and cells were exposed to LPS of Escherichia coli (E. coli) (10 μg mL ) for 24, 48, and 72 h. The cells were subjected to specific parameters of phototherapy (PT) (LASERTable-InGaAsP-780 ± 3 nm, 25 mW, 3 J cm ). Cell proliferation (alamarBlue ), viability (Trypan Blue) and synthesis of CCL2 (ELISA) were evaluated. Data were statistically analyzed by the Kruskal-Wallis and Mann-Whitney test (α = 5%). Proliferation and viability of both cell lines decreased after LPS treatment at 48 and 72 h. Enhanced synthesis of CCL2 by gingival fibroblasts occurred at 24 h, while epithelial cells increased synthesis of this chemokine at 48 and 72 h. PBM enhanced cell proliferation and viability in a time-dependent manner for both cell lines exposed or not to LPS, while synthesis of CCL2 by cells exposed to PT decreased over time. PBM caused biomodulatory effects on gingival fibroblasts and epithelial cells previously treated with LPS. These effects may decrease tissue inflammatory response and accelerate wound healing of oral mucosal tissue.

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The hepatocyte growth factor-expressing character is required for mesenchymal stem cells to protect the lung injured by lipopolysaccharide in vivo.

Acute respiratory distress syndrome (ARDS) is a life-threatening condition in critically ill patients. Recently, we have found that mesenchymal stem cells (MSC) improved the permeability of human lung microvascular endothelial cells by secreting hepatocyte growth factor (HGF) in vitro. However, the properties and functions of MSC may change under complex circumstances in vivo. Here, we sought to determine the role of the HGF-expressing character of MSC in the therapeutic effects of MSC on ARDS in vivo.

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Interaction between mesenchymal stem cells and endothelial cells restores endothelial permeability via paracrine hepatocyte growth factor in vitro.

Mesenchymal stem cells (MSCs) have potent stabilising effects on vascular endothelium injury, inhibiting endothelial permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secrete more factors by MSC-endothelial cell (MSC-EC) interactions. We hypothesised that MSC-EC interactions restore endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF.

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Conditioned medium from umbilical cord mesenchymal stem cells induces migration and angiogenesis.

Umbilical cord mesenchymal stem cells (UC-MSCs) have been suggested as a candidate for various clinical applications, however, major limitations include the lack of organ-specific accumulation and low survival rates of transplanted cells. In the present study, it was hypothesized that the paracrine effects of UC‑MSCs may enhance stem cell-based tissue repair and regeneration by promoting the specific homing of stem/progenitor cells and the overall ability to drive them to the damaged area. UC-MSCs-derived conditioned medium (UC-CM) was analyzed using liquid chip and ELISA techniques. In vitro tube formation assays of human umbilical vein endothelial cells (HUVECs) and UC-MSCs were then performed to assess the angiogenic properties of UC-CM. Subsequently, UC-MSCs, HUVECs and fibroblasts were labeled with PKH26 for an in vivo cell migration assay. The expression levels of C-X-C chemokine receptor 4 (CXCR4), C-C chemokine receptor 2 (CCR2) and c-met were determined in the UC-MSCs, HUVECs and fibroblasts using reverse transcription-quantitative polymerase chain reaction and flow cytometry. UC-CM was incubated with or without antibodies, and the contribution of stromal cell-derived factor 1 (SDF-1), monocyte chemotactic protein 1 (MCP-1) and hepatocyte growth factor (HGF) on the migration of cells was investigated in vitro. The results demonstrated that UC-MSCs secreted different cytokines and chemokines, including increased quantities of SDF-1, MCP-1 and HGF, in addition to the angiogenic factors, vascular cell adhesion protein-1, interleukin-8, insulin-like growth factor-1 and vascular endothelial growth factor. The total lengths of the tubes were significantly increased in the UC-MSCs and HUVECs incubated in UC-CM compared with those incubated in Dulbecco's modified Eagle's medium. In vivo cell migration assays demonstrated that UC-CM was a chemotactic stimulus for the UC-MSCs and HUVECs. In vitro Matrigel migration and scratch healing assays demonstrated that UC-CM increased the migration of CXCR4-positive or/and CCR2-positive cells in a dose-dependent manner. In addition, different molecules were screened under antibody-based blocking migration conditions. The data revealed that the SDF-1/CXCR4 and MCP-1/CCR2 axes were involved in the chemoattractive activity of UC-CM and suggested that the effective paracrine factor of UC-CM is a large complex rather than a single factor. The results of the present study supported the hypothesis that UC-MSCs release soluble factors, which may extend the therapeutic applicability of stem cells.

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Potential of a cryopreserved cultured dermal substitute composed of hyaluronic acid and collagen to release angiogenic cytokine.

An allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a spongy matrix of hyaluronic acid (HA) and collagen (Col), which was then cryopreserved. This cryopreserved allogeneic CDS (CDS-1; cryopreserved for 1 month, CDS-6; cryopreserved for 6 months) was thawed and re-cultured for a period of 7 days to investigate the potential of the CDS for wound treatment. The cell metabolic activity in the CDS and their cytokine production were measured using an MTT assay and ELISA. Fibroblast metabolic activity in each CDS-1 and CDS-6 immediately after thawing and following 3 and 7 days of re- cultivation was 56, 67 and 93%, and 49, 64 and 86%, respectively, of that before cryopreservation. The amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released from the CDS-1 on days 1, 3 and 7 of re-cultivation after thawing was 8, 44 and 92% (VEGF) and 3, 7 and 28% (HGF), respectively, of that before cryopreservation. The amount of VEGF and HGF released from the CDS-6 on days 1, 3 and 7 of re-cultivation after thawing was 9, 32 and 45% (VEGF) and 6, 10 and 27% (HGF), respectively, of that before cryopreservation. These findings showed that the potential of the CDS was restored to some extent over the first 3 days of re-cultivation after thawing. The potential of the CDS for wound treatment was then evaluated using a wound surface model, in which the each CDS-1 and CDS-6 that was re-cultured for 3 days after thawing was elevated at the air/culture medium interface, and a wound dressing was placed on top, and then cultured for 5 days. Two different types of wound dressing were tested. Fibroblasts in the CDS in Group II (placing a wound dressing with EGF) released increased amount of VEGF and HGF compared with that in Group I (placing a wound dressing without EGF). These findings suggest that re-culture of the CDS for 3 days following thawing results in a CDS with improved wound healing potential and that an EGF-incorporating wound dressing is useful as a top dressing for the CDS.

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The HGF/c-Met axis synergizes with G-CSF in the mobilization of hematopoietic stem/progenitor cells.

As granulocyte-colony-stimulating factor (G-CSF)-induced mobilization of hematopoietic stem/progenitor cells (HSPCs) increases human serum levels of hepatocyte growth factor (HGF), our aim was to investigate the role of HGF and its receptor, c-Met, in the mobilization of HSPC. CD34(+) cells and leukocytes were isolated from the bone marrow (BM) of normal donors and the peripheral blood (PB) of patients mobilized with G-CSF and chemotherapy. Plasma HGF levels were evaluated by ELISA and HGF and c-Met expression by RT-PCR, fluorescence-activated cell sorter (FACS) analysis, and confocal microscopy. Because matrix metalloproteinases (MMPs) facilitate migration across extracellular matrix (ECM) and basement membranes, we also examined expression of MMP-9 and membrane type 1 (MT1)-MMP in hematopoietic cells after HGF stimulation. We found that plasma HGF levels in mobilized (m)PB were higher in patients who are good mobilizers and correlated with their white blood cell (WBC) and CD34(+) cell counts. Moreover, HGF and c-Met expression was significantly higher in mPB CD34(+) cells and leukocytes than in their steady-state BM counterpart cells and was up-regulated by G-CSF. Like G-CSF, HGF increased the secretion of MMP-9 and the expression of MT1-MMP in leukocytes, which was abrogated by the c-Met inhibitor K-252a. This inhibitor also significantly reduced the trans-Matrigel migration of mPB CD34(+) cells toward HGF. Our results suggest that G-CSF-mediated HSPC mobilization occurs in part through the HGF/c-Met axis in HSPC and myeloid cells, eliciting increased production of matrix-degrading enzymes and subsequently facilitating egress of HSPC.

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Increased number of mesenchymal stem cell-like cells in peripheral blood of patients with bone sarcomas.

The number of peripheral blood mesenchymal stem cells (PBMSCs) may increase under pathological conditions. We sought to compare the number of MSC-like cells in the peripheral blood of patients with bone sarcomas with healthy controls and to analyze related cytokines in the peripheral blood plasma.

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Peritoneal fluid and serum levels of hepatocyte growth factor may predict the activity of endometriosis.

The suitable parameter in PF as well as in serum that may predict the activity of endometriosis is not well described. Therefore, we tried to examine the peritoneal fluid (PF) and serum concentrations of hepatocyte growth factor (HGF) in different revised American Society of Reproductive Medicine (r-ASRM) staging and morphologic appearances of endometriosis in an attempt to determine whether HGF can be clinically useful to predict the activity of pelvic endometriosis.

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Plasminogen- and colony-stimulating factor-1-associated markers in bladder carcinoma: diagnostic value of urokinase plasminogen activator receptor and plasminogen activator inhibitor type-2 using immunocytochemical analysis.

The expression of plasminogen- and colony-stimulating factor-1-associated markers was first investigated in seven bladder carcinoma cell lines and in 15 primary bladder tumors using RT-PCR (mRNAs), zymography (protein activity), ELISA and immunocytochemistry analysis (ICC) (protein levels). The mRNAs expression, the activity and the levels of the secreted proteins were not informative. Only urokinase plasminogen activator receptor (uPA-R/CD87) and possibly plasminogen activator inhibitor type-2 (PAI2) antigen expression at the cellular levels seem to be useful markers. uPA-R antigen expression correlated with the secretion of hepatocyte growth factor (HGF) ( P=0.016) and the motility of the bladder tumor cells ( P=0.014), two markers associated with a poor prognosis in bladder carcinoma. To validate our technique and confirm these preliminary results, uPA-R and PAI2 antigen expression was determined in the imprints from 129 resected bladder carcinoma fragments. uPA-R correlated with the grade ( P=0.002), tumor invasion ( P=0.003) and the ploidy ( P=0.05) of the bladder carcinomas and with the low overall survival ( P=0.045) of the patients. PAI2 correlated only with the stage ( P=0.02) and low overall survival ( P=0.038). We conclude that in bladder carcinomas, studying the transcripts of PAs, PAIs, CSF-1 and its receptor, as well as measuring their concentration or activity in culture supernatants was of no clinical interest in terms of diagnostic or prognostic value. Only the ICC of uPA-R, which correlated with the major histopathological parameters of tumors and the low overall survival, proved to be a diagnostic and prognostic marker.

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