Endothelial Cell Migration on Poly(ε-caprolactone) Nanofibers Coated with a Nanohybrid Shish-Kebab Structure Mimicking Collagen Fibrils.

Regulating cell migration dynamics is of significance in tissue engineering and regenerative medicine. A 3D scaffold was created to provide various topographies based on a poly(ε-caprolactone) (PCL) self-induced nanohybrid shish-kebab structure, which consisted of aligned PCL nanofibers and spaced PCL crystal lamellae grown on the fibers. Electrospinning was applied followed by self-induced crystallization. The results resembled natural collagen fibrils in an extracellular matrix. This variable microstructure enabled control of cell adhesion and migration. The kebab size was controlled by initial PCL concentrations. The geometry of cells seeded on the fibers was less elongated, but the adhesion was more polarized with a higher nuclear shape index and faster migration speed. These results could aid in rapid endothelialization in tissue engineering.

LPS-induced autophagy in human dental pulp cells is associated with p38.

Lipopolysaccharides (LPS), which are components of the cell wall of Gram-negative bacteria, are among the important factors that induce inflammation, including pulpitis. Autophagy in human dental pulp cells (hDPCs) acts as a protective mechanism that promotes cell survival under adverse conditions through different signaling pathways. In this study, we examined whether LPS increases autophagy in hDPCs and investigated the role of mitogen-activated protein kinases signaling and nuclear factor κB (NF-κB) in this process. We found that stimulation of hDPCs with 0.1 µg/mL LPS increased the protein and mRNA levels of autophagy markers, beclin1 and microtubule associated protein light chain 3II (LC3II). In addition, acridine orange staining and transmission electron microscopy demonstrated the induction of autophagy upon the treatment of LPS. Furthermore, LPS affected phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and the nuclear translocation of NF-κB. While p38 inhibitor suppressed the LPS-induced increase in protein levels of beclin1 and LC3-II. Our results suggest that LPS induced autophagy in hDPCs and affected the phosphorylation of p38, ERK, and JNK, as well as the nuclear translocation of NF-κB. Phosphorylation of p38 may be involved in LPS-induced autophagy in hDPCs.

Biologically responsive, long-term release nanocoating on an electrospun scaffold for vascular endothelialization and anticoagulation.

A critical challenge to the development of tissue engineering small-diameter vascular grafts is to achieve rapid endothelialization and long-term anticoagulation. It is necessary to graft both adhesion and antithrombus factors onto the surface of polycaprolactone without burst release to promote endothelial cell affinity and antithrombogenicity. A bionic structure with a nanocoating that allows a biologically responsive, long-term release was employed in this work to enable the grafting of various bioactive molecules such as gelatin, polylysine, and heparin. This approach involved orienting the biomimetic vascular structures; the self-assembly grafting of gelatin, polylysine, and heparin nanoparticles; and genipin crosslinking to form a multiphase crosslinked nanocoating. In this biologically inspired design, vascular endothelialization and long-term anticoagulation were successfully induced through a matrix metallopeptidase 2 regulative mechanism by delivering both adhesion and antithrombus factors with a responsive, long-term release without burst release. The method provided a simple and effective approach for delivering dual factors for tissue engineering small-diameter vascular grafts.

Expression of Different Isoforms of Versican During the Development of Mouse Mandibular First Molars.

Versican is a large chondroitin sulfate proteoglycan enriched in the extracellular matrix, and it has at least four different isoforms, termed V0, V1, V2, and V3. Although several studies have demonstrated that versican is stably expressed in various developing organs, the expression of versican isoforms during tooth development has not been elucidated yet. Therefore, the present study was to investigate the expression of versican isoforms in the developing mouse molars. The mandibular first molars from embryonic day (E) 11.5 to postnatal day (PN) 21 were used to investigate the expression of versican isoforms by immunohistochemistry, and the gene expressions of versican (Vcan) isoforms from E13.5 to PN7 were analyzed by quantitative real-time PCR. The results exhibited different expressing patterns of versican isoforms-the stellate reticulum (SR) and the dental mesenchymal cells adjacent to Hertwig's Epithelial Root Sheath (HERS) only expressed V1 and the mature odontoblasts mainly expressed V2, while the dental papilla and the ameloblasts might both express V0/V1/V2. These results suggested that different versican isoforms may act different roles in the tooth development, and we speculated that V0/V1 might be intimately involved in the cell proliferation while V2 was associated in the cytodifferentiation.

Effects of aligned and random fibers with different diameter on cell behaviors.

Tissue-engineered vascular grafts show promise as a treatment for patients with diseased or damaged arteries. The formation of a confluent endothelium lumen on scaffold is one of the key issues in vascular tissue engineering. The endothelial layer is composed of endothelial cells (ECs) aligned along the long axis of the vessel. The contact guidance effect is believed to fundamentally impact cell morphology, alignment, and behavior. By tuning the scaffold topography where the EC cells reside, the cell adhesion, shape, and other behaviors might be regulated. To this end, poly(ε-caprolactone) was electrospun to obtain different topographies by varying fiber diameter and orientation. The morphology and alignment of EC cells were investigated by analyzing fluorescence staining images. The results show that nano-sized fibers significantly enhance cell adhesion and proliferation due to the comparable geometric feature size of the fiber diameter. ECs align along the fiber oriented direction for both microfibers and nanofibers. It shows a better alignment of ECs on micro-aligned fibers than on nano-aligned fibers, which is due to the active size range of the contact guidance effect. These results indicate that scaffolds with ordered topographies might favor the formation of a confluent endothelium for vascular tissue engineering.

Lipopolysaccharide stimulation improves the odontoblastic differentiation of human dental pulp cells.

Lipopolysaccharide (LPS) is one of the causative agents of pulpitis and previous studies have demonstrated that the LPS stimulation of human aortic valve interstitial cells induces inflammatory mediators and the gene expression of osteogenic factors. Therefore, in the present study, it was hypothesized that LPS affects the odontoblastic differentiation of human dental pulp cells (hDPCs). In order to investigate this, an in vitro study using hDPCs was performed. Increased alkaline phosphatase (ALP) activity was observed in the hDPCs treated with LPS, which was more marked when the cells were costimulated with odontogenic induction medium (OM). LPS also appeared to increase the gene expression levels of dentin sialophosphoprotein and dentin matrix protein­1 and the protein expression level of dental sialoprotein in the hDPCs, particularly in combination with OM. In addition, the size and the number of nodules formed in the hDPCs exposed to OM and LPS were increased compared to those stimulated by OM alone. To determine the role of nuclear factor κB (NF­κB) during the LPS­induced odontoblastic differentiation of hDPCs, immunofluorescence was performed. The nuclear translocation of NF­κB, induced by LPS was confirmed, suggesting its involvement in the LPS­induced increase in odontoblastic differentiation of hDPCs. In conclusion, there may be an association between LPS stimulation, with or without OM, and odontoblastic differentiation.

Expression and function of the actin-severing protein adseverin in the proliferation, migration, and differentiation of dental pulp cells.

INTRODUCTION: Adseverin is an actin-severing and actin-capping protein that is primarily expressed in secretory cells, where it regulates the filamentous actin cytoskeleton during cell differentiation and exocytosis. However, little is known regarding its regulatory role in dental pulp cells (DPCs). This study examined the expression and function of adseverin in the proliferation, migration, and odontoblastic differentiation of DPCs. METHODS: DPCs were assayed for morphologic changes, proliferation, migration, alkaline phosphatase activity, and dentin sialoprotein and dentin matrix protein-1 protein levels in vitro after knockdown of adseverin by using small interfering RNA. Tooth germs isolated from Sprague-Dawley rats were processed for immunohistochemistry analysis of adseverin. RESULTS: Adseverin expression was increased in a time-dependent fashion in the early stage of odontoblastic differentiation. When adseverin expression was suppressed in DPCs, their cellular morphology was altered, and their proliferation, migration, and odontoblastic differentiation were substantially decreased in vitro. Secretory odontoblasts in the tooth germ at day 5 post partum expressed a stronger adseverin signal compared with those at days 1 and 3 post partum. CONCLUSIONS: Adseverin may play a crucial role in the proliferation, migration, and odontoblastic differentiation of DPCs via filamentous actin cytoskeleton regulation. However, further investigations are required to clarify the underlying mechanisms.

Isolation and characterization of a gene encoding a polyethylene glycol-induced cysteine protease in common wheat.

Plant cysteine protease (CP) genes are induced by abiotic stresses such as drought, yet their functions remain largely unknown. We isolated the full-length cDNA encoding a Triticum aestivum CP gene, designated TaCP, from wheat by the rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that TaCP contains an open reading frame encoding a protein of 362 amino acids, which is 96% identical to barley cysteine protease HvSF42. The TaCP transcript level in wheat seedlings was upregulated during polyethylene glycol (PEG) stress, with a peak appearing around 12 h after treatment. TaCP expression level increased rapidly with NaCl treatment at 48 h. TaCP responded strongly to low temperature (4 degree C) treatment from 1 h post-treatment and reached a peak of about 40-fold at 72 h. However, it showed only a very slight response to abscisic acid (ABA). More than one copy of TaCP was present in each of the three genomes of hexaploid wheat and its diploid donors. TaCP fused with green fluorescent protein (GFP) was located in the plasma membrane of onion epidermis cells. Transgenic Arabidopsis plants overexpressing TaCP showed stronger drought tolerance and higher CP activity under water-stressed conditions than wild-type Arabidopsis plants. The results suggest that TaCP plays a role in tolerance to water deficit.