FERMT2 upregulation in CAFs enhances EMT of OSCC and M2 macrophage polarization.

OBJECTIVES: FERMT2 upregulation was associated with malignant tumor behaviors, including epithelial-to-mesenchymal (EMT). This study aimed to characterize the expression profile of FERMT2 in oral squamous cell carcinoma (OSCC) and to explore its involvement in the tumor microenvironment sculptured by oral cancer-associated fibroblasts (OCAFs). MATERIALS: Previous bulk-seq (TCGA-HNSC) and single-cell RNA-seq data sets were retrieved for bioinformatic analysis. Human OSCC lines SCC15 and CAL27, primary normal oral fibroblasts (NOFs), OCAFs, and THP-1 cells were used for intro studies. RESULTS: FERMT2 expression was significantly higher in CAFs compared with OSCC tumor cells and normal fibroblasts. Higher FERMT2 expression might independently predict unfavorable disease-specific survival (DSS) in patients with OSCC. Knockdown of FERMT2 suppressed the expression and secretion of IGFBP7, SPARC, TIMP3, COL4A1, and IGFBP4 in OCAFs. OCAFs with FERMT2 knockdown had significantly weakened capability to induce the invasion of OSCC cells and the expression of mesenchymal markers. FERMT2 knockdown impaired the inducing effect of OCAFs on the migration of M0 macrophages and the expression of M2 macrophage markers. CONCLUSIONS: FERMT2 could modulate the production and secretion of IGFBP7, SPARC, COL4A1, and IGFBP4 in OCAFs, thereby inducing the EMT of OSCC and M2 macrophage polarization.

Possible Mechanism of Placental Mesenchymal Stem Cell-Derived Neural Cell Transplantation on the Recovery of Neurogenic Bladder Function after Spinal Cord Injury.

After spinal cord injury, more neurogenic bladder function is caused. The purpose of this article is to investigate the possible mechanism of placental mesenchymal stem cell-derived neural cell transplantation on the recovery of neurogenic bladder function after spinal cord injury. 50 SPF Wistar rats were selected to establish a spinal cord injury rat model and divided into experimental groups and in the control group, 25 animals in each group, the experimental group was transplanted with placental mesenchymal stem cell-derived nerve cells, and the urodynamics and TUNEL positive rate were compared. The results of the study showed that compared with the control group, the maximum bladder capacity and bladder compliance of the experimental group increased significantly (P<0.01), and the bladder basic pressure and urinary leakage pressure decreased (P<0.05). The values of these four items are 2.318ml, 28.892cm H2O, 46.34cm H2O, and 0.1389ml/cm H2O, respectively. It can be seen that the transplantation of neural cells derived from placental mesenchymal stem cells is of great significance for the recovery of neurogenic bladder function after spinal cord injury.

Hydroxyapatite/NELL-1 Nanoparticles Electrospun Fibers for Osteoinduction in Bone Tissue Engineering Application.

BACKGROUND: As commonly bone defect is a disease of jaw that can seriously affect implant restoration, the bioactive scaffold can be used as potential systems to provide effective repair for bone defect. PURPOSE: A osteoinductive bone tissue engineering scaffold has been prepared in order to explore the effect of bioactive materials on bone tissue engineering. METHODS: In this study, NELL-1 nanoparticles (Chi/NNP) and nano hydroxyapatite were incorporated in composite scaffolds by electrospinning and characterized using TEM, SEM, contact angle, tensile tests and in vitro drug release. In vitro biological activities such as MC3T3-E1 cell attachment, proliferation and osteogenic activity were studied. RESULTS: With the addition of nHA and nanoparticles, the fiber diameter of PCL/BNPs group, PCL/NNPs group and PCL/nHA/NNPs group was significantly increased. Moreover, the hydrophilic hydroxyl group and amino group presented in nHA and nanoparticles had improved the hydrophilicity of the composite fibers. The composite electrospun containing Chi/NNPs can form a double protective barrier which can effectively prolong the release time of NELL-1 growth factor. In addition, the hydroxyapatite/NELL-1 nanoparticles electrospun fibers can promote attachment, proliferation, differentiation of MC3T3-E1 cells and good cytocompatibility, indicating better ability of inducing osteogenic differentiation. CONCLUSION: A multi-functional PCL/nHA/NNPs composite fiber with long-term bioactivity and osteoinductivity was successfully prepared by electrospinning. This potential composite could be used as scaffolds in bone tissue engineering application after in vivo studies.

The effects of Sema3A overexpression on the proliferation and differentiation of rat gingival mesenchymal stem cells in the LPS-induced inflammatory environment.

Semaphorin3A has been identified as a potent osteoprotective factor that simultaneously inhibits bone resorption and promotes bone formation. The present study demonstrates the effect of the overexpression of Sema3A on the proliferation and differentiation of rat gingival mesenchymal stem cells (GMSCs) in the lipopolysaccharide (LPS)-induced inflammatory environment. rGMSCs were transfected with viral stocks of pLenO-GTP-Sema3A (Lv-Sema3A group) or pLenO-GTP (Lv-NC group), with rGMSCs as a control. The transfection efficiency was determined by flow cytometry. Cell proliferation was assessed using a Cell Counting Kit-8 assay. The expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2) were determined at 3, 7 and 14 days after the osteogenic induction culture with or without LPS using real-time PCR and Western blot. Alizarin Red staining was performed at 28 days. A pLenO-GTP-Sema3A-mediated transfection of rGMSC stably overexpressing Sema3A was built up. The overexpression of Sema3A promoted cell proliferation in the LPS-induced inflammatory environment. In addition, osteogenesis-related genes were upregulated in the Lv-Sema3A group compared with the control group. Also, after LPS administration, the overexpression of Sema3A enhanced the expression of the osteogenic genes in the LPS-induced inflammatory environment. Hence, Sema3A gene-modified rGMSCs show better osteogenic differentiation and proliferation capacities compared with rGMSCs in the LPS-induced inflammatory environment.

Chitosan-stablized bovine serum albumin nanoparticles having ability to control the release of NELL-1 protein.

The study was designed to prepare and evaluate chitosan stabilized-albumin nanoparticles as NELL-1 protein carriers(Chi/NNPs). The Chi/NNPs were prepared by desolvation method and then stabilized by chitosan through electrostatic interaction. The Chi/NNPs were characterized for drug loading efficiency, surface morphology, particle size, surface charge. Fluorescein isothiocyanate-labeled chitosan was used to confirm the homogeneity of chitosan coating on the BSA nanoparticles. The NELL-1 bioactivity of Chi/NNPs and the release kinetics were investigated in vitro. It was observed that the mean particle size with chitosan (0.075 wt%,0.15 wt%, 0.3 wt%, respectively) and the surface charge were 368.663 ±â€¯15.470 nm, 382.881 ±â€¯18.767 nm, 390.480 ±â€¯11.465 nm and +25.03 ±â€¯1.42 mV, +30.27 ±â€¯1.80 mV, +31.03 ±â€¯2.05 mV respectively. Drug entrapment efficiency ranged from 87.83% to 89.30%. The Chi/NNPs prepared with the 0.15 wt% chitosan were able to successfully control the release of NELL-1 and maintain a sustained release for up to 8 days. Furthermore, more than 82.67 ±â€¯8.74% of the loaded protein's bioactivity was preserved in Chi/NNPs over the period of the investigation. Our findings suggest that Chi/NNPs as promising protein delivery nanocarriers have the ability to maintain sustained release kinetics and to preserve the bioactivity of released NELL-1.

[Effects of cofilin phosphorylation on the actin cytoskeleton reorganization induced by shear stress].

OBJECTIVE: To explore the effects of cofilin on the actin cytoskeleton reorganization in osteoblasts induced by fluid shear stress. METHODS: Fluid shear stress (1.2 Pa) was applied to osteoblasts for 0 (control group), 15, 30, 45, 60, 120 min in vitro. Cells were stained with fluorescein isothiocyanate (FITC)-phalloidin for fiber-actin, and confocal laser scanning microscope(CLSM) was used to observe the fluorescence of fiber-actin. Western blotting was used to detect the expression of the cofilin and the phospho-cofilin. RESULTS: Actin filaments became organized into stress fibers that were thicker and more abundant than those in non-flowed cells. The fluorescence intensity (38.00 ± 6.88) of fiber-actin after 120 min (42.93 ± 6.41) loading it was 2.8 times as much as that in control group (15.41 ± 3.60, P < 0.05). Additionally, the level of phospho-cofilin protein was dramatically elevated after loading. Fluid shear stress induced an initial decrease of cofilin at 60 min. However, at 120 min cofilin (0.254 ± 0.026) increased to 1.5 times as much as that at 60 min (0.162 ± 0.004). CONCLUSIONS: The results indicate that cofilin phosphorylation mediates fiber-actin reorganization in the osteoblasts induced by fluid shear stress.

[Effect of cytoskeleton integrity on the expression of c-fos in osteoblasts induced by fluid shear stress].

OBJECTIVE: To investigate the effect of cytoskeleton integrity on the expression of c-fos gene in osteoblasts induced by fluid shear stress. METHODS: BALB/c mouse primary osteoblasts were divided into four groups (according to fluid shear stress loaded or not and cytochalasin D used or not). The Tagman probe real-time PCR and immunofluorescence were performed to detect the expression levels of c-fos mRNA, c-fos protein and cytoskeleton, respectively. The data were analysed using two-way ANOVA. RESULTS: In control group and cytochalasin D group, fluid shear stress could significantly increase the expression levels of c-fos mRNA (0.1637 +/- 0.0303 and 0.0104 +/- 0.0070, respectively) and protein (177.14 +/- 9.37 and 150.95 +/- 6.17, respectively) in osteoblasts, compared with the unloaded osteoblasts of the control group and the cytochalasin D group (0.0057 +/- 0.0021 and 0.0032 +/- 0.0014, respectively for c-fos mRNA, and 117.96 +/- 4.11 and 119.77 +/- 5.19, respectively for protein, P < 0.05). Induced by the fluid shear stress, the expression levels of c-fos mRNA and protein in cytochalasin D group were lower than control group, and the difference had statistical significance (P < 0.05). CONCLUSIONS: The cytoskeleton integrity in osteoblasts was essential to the expression of c-fos gene induced by fluid shear stress.

[Effects of LIMK2 RNA interference on the mechanosensitivity of c-fos gene in osteoblast].

OBJECTIVE: To study the effects of cytoskeleton reorganization inhibition with LIMK2 RNAi upon the mechanosensitivity of c-fos gene in osteoblast. METHODS: Mouse primary osteoblast was treated with LIMK2 specific siRNA (RNAi Group), negative control siRNA (NC Group), and then were loaded or unloaded by fluid shear stress. Real-time PCR and immunofluorescence were used to detect the c-fos expression levels and statistics analysis was performed. RESULTS: When the cytoskeleton reorganization was inhibited with RNAi only, the c-fos mRNA (0.0108 +/- 0.0074 and 0.0042 +/- 0.0018, t = -1.86, P > 0.05) and protein (121 +/- 7 and 119 +/- 6, t = -1.272, P > 0.05) expression levels of each unloaded group had no significant difference; Fluid shear stress could up-regulate the c-fos mRNA (0.2203 +/- 0.1532 vs 0.0042 +/- 0.0018, t = -707.35, P < 0.05)and protein (178 +/- 12 vs 119 +/- 6, t = -30.761, P < 0.05) expression; After the cytoskeleton reorganization was inhibited with RNAi, the c-fos mRNA (0.5280 +/- 0.0879 vs 0.2203 +/- 0.1532, t = -1007.00, P < 0.05) and protein (224 +/- 46 vs 178 +/- 12, t = -6.853, P < 0.05) expression induced by fluid shear stress had significant difference. Cytoskeleton reorganization inhibition with RNAi had synergistic effect upon the expression of c-fos mRNA (F = 84.388, P < 0.05) and protein (F = 42.409, P < 0.05) induced by fluid shear stress. CONCLUSION: Using RNAi against LIMK2 to inhibit the cytoskeleton reorganization can promote the expression of c-fos gene and thus enhance the mechanosensitivity of c-fos gene in osteoblast.