Biosynthesis and Function of VIP and Oxytocin: Mechanisms of C-terminal Amidation, Oxytocin Secretion and Transport.

In this review, we provide the status of research on vasoactive intestinal peptide (VIP) and oxytocin, typical C-terminal α-amidated peptide hormones, including their precursor protein structures, processing and C-terminal α-amidation, and the recently identified mechanisms of regulation of oxytocin secretion and its transportation through the blood brain barrier. More than half of neural and endocrine peptides, such as VIP and oxytocin, have the α-amide structure at their C-terminus, which is essential for biological activities. We have studied the synthesis and function of C-terminal α-amidated peptides, including VIP and oxytocin, since the 1980s. Human VIP mRNA encoded not only VIP but also another related C-terminal α-amidated peptide, PHM-27 (peptide having amino-terminal histidine, carboxy-terminal methionine amide, and 27 amino acid residues). The human VIP/PHM-27 gene is composed of 7 exons and regulated synergistically by cyclic AMP and protein kinase C pathways. VIP has an essential role in glycemic control using transgenic mouse technology. The peptide C-terminal α-amidation proceeded through a 2-step mechanism catalyzed by 2 different enzymes encoded in a single mRNA. In the oxytocin secretion from the hypothalamus/the posterior pituitary, the CD38-cyclic ADP-ribose signal system, which was first established in the insulin secretion from pancreatic ß cells of the islets of Langerhans, was found to be essential. A possible mechanism involving RAGE (receptor for advanced glycation end-products) of the oxytocin transportation from the blood stream into the brain through the blood-brain barrier has also been suggested.

Transthyretin Is Commonly Upregulated in the Hippocampus of Two Stress-Induced Depression Mouse Models.

Chronic stress can affect gene expression in the hippocampus, which alters neural and cerebrovascular functions, thereby contributing to the development of mental disorders such as depression. Although several differentially expressed genes in the depressed brain have been reported, gene expression changes in the stressed brain remain underexplored. Therefore, this study examines hippocampal gene expression in two mouse models of depression induced by forced swim stress (FSS) and repeated social defeat stress (R-SDS). Transthyretin (Ttr) was commonly upregulated in the hippocampus of both mouse models, as determined by microarray, RT-qPCR, and Western blot analyses. Evaluation of the effects of overexpressed Ttr in the hippocampus using adeno-associated virus-mediated gene transfer revealed that TTR overexpression induced depression-like behavior and upregulation of Lcn2 and several proinflammatory genes (Icam1 and Vcam1) in the hippocampus. Upregulation of these inflammation-related genes was confirmed in the hippocampus obtained from mice vulnerable to R-SDS. These results suggest that chronic stress upregulates Ttr expression in the hippocampus and that Ttr upregulation may be involved in the induction of depression-like behavior.

EPAC2 acts as a negative regulator in Matrigel-driven tubulogenesis of human microvascular endothelial cells.

Angiogenesis is physiologically essential for embryogenesis and development and reinitiated in adult animals during tissue growth and repair. Forming new vessels from the walls of existing vessels occurs as a multistep process coordinated by sprouting, branching, and a new lumenized network formation. However, little is known regarding the molecular mechanisms that form new tubular structures, especially molecules regulating the proper network density of newly formed capillaries. This study conducted microarray analyses in human primary microvascular endothelial cells (HMVECs) plated on Matrigel. The RAPGEF4 gene that encodes exchange proteins directly activated by cAMP 2 (EPAC2) proteins was increased in Matrigel-driven tubulogenesis. Tube formation was suppressed by the overexpression of EPAC2 and enhanced by EPAC2 knockdown in endothelial cells. Endothelial cell morphology was changed to round cell morphology by EPAC2 overexpression, while EPAC2 knockdown showed an elongated cell shape with filopodia-like protrusions. Furthermore, increased EPAC2 inhibited endothelial cell migration, and ablation of EPAC2 inversely enhanced cell mobility. These results suggest that EPAC2 affects the morphology and migration of microvascular endothelial cells and is involved in the termination and proper network formation of vascular tubes.

Emerging Role of AP-1 Transcription Factor JunB in Angiogenesis and Vascular Development.

Blood vessels are essential for the formation and maintenance of almost all functional tissues. They play fundamental roles in the supply of oxygen and nutrition, as well as development and morphogenesis. Vascular endothelial cells are the main factor in blood vessel formation. Recently, research findings showed heterogeneity in vascular endothelial cells in different tissue/organs. Endothelial cells alter their gene expressions depending on their cell fate or angiogenic states of vascular development in normal and pathological processes. Studies on gene regulation in endothelial cells demonstrated that the activator protein 1 (AP-1) transcription factors are implicated in angiogenesis and vascular development. In particular, it has been revealed that JunB (a member of the AP-1 transcription factor family) is transiently induced in endothelial cells at the angiogenic frontier and controls them on tip cells specification during vascular development. Moreover, JunB plays a role in tissue-specific vascular maturation processes during neurovascular interaction in mouse embryonic skin and retina vasculatures. Thus, JunB appears to be a new angiogenic factor that induces endothelial cell migration and sprouting particularly in neurovascular interaction during vascular development. In this review, we discuss the recently identified role of JunB in endothelial cells and blood vessel formation.

Metformin Mitigates DPP-4 Inhibitor-Induced Breast Cancer Metastasis via Suppression of mTOR Signaling.

The biological influence of antidiabetic drugs on cancer cells and diabetic cancer patients has not yet been completely elucidated. We reported that a dipeptidyl peptidase (DPP)-4 inhibitor accelerates mammary cancer metastasis by inducing epithelial-mesenchymal transition (EMT) through the CXCL12/CXCR4/mTOR axis. Metformin has been shown to inhibit the mTOR signaling pathway. In this study, we investigated whether metformin mitigates breast cancer metastasis induced by a DPP-4 inhibitor via suppression of mTOR signaling. In cultured mouse mammary and human breast cancer cells, metformin suppressed DPP-4 inhibitor KR62436 (KR)-induced EMT and cell migration via suppression of the mTOR pathway associated with AMPK activation. For the in vivo study, metformin intervention was performed in an allograft 4T1 breast cancer model mouse with or without KR. We also analyzed mice transplanted with shRNA-mediated DPP-4 knockdown 4T1 cells. Treatment with metformin inhibited the lung metastasis of DPP-4-deficient 4T1 mammary tumor cells generated by either KR administration or DPP-4 knockdown. Immunostaining of primary tumors indicated that DPP-4 suppression promoted the expression of EMT-inducing transcription factor Snail through activation of the CXCR4-mediated mTOR/p70S6K pathway in an allograft breast cancer model; metformin abolished this alteration. Metformin treatment did not alter DPP-4-deficiency-induced expression of CXCL12 in either plasma or primary tumors. Our findings suggest that metformin may serve as an antimetastatic agent by mitigating the undesirable effects of DPP-4 inhibitors in patients with certain cancers. IMPLICATIONS: Metformin could combat the detrimental effects of DPP-4 inhibitor on breast cancer metastasis via mTOR suppression, suggesting the potential clinical relevance. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/1/61/F1.large.jpg.

Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.

Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.

Role of Arginine Methylation in Alternative Polyadenylation of VEGFR-1 (Flt-1) pre-mRNA.

Mature mRNA is generated by the 3' end cleavage and polyadenylation of its precursor pre-mRNA. Eukaryotic genes frequently have multiple polyadenylation sites, resulting in mRNA isoforms with different 3'-UTR lengths that often encode different C-terminal amino acid sequences. It is well-known that this form of post-transcriptional modification, termed alternative polyadenylation, can affect mRNA stability, localization, translation, and nuclear export. We focus on the alternative polyadenylation of pre-mRNA for vascular endothelial growth factor receptor-1 (VEGFR-1), the receptor for VEGF. VEGFR-1 is a transmembrane protein with a tyrosine kinase in the intracellular region. Secreted forms of VEGFR-1 (sVEGFR-1) are also produced from the same gene by alternative polyadenylation, and sVEGFR-1 has a function opposite to that of VEGFR-1 because it acts as a decoy receptor for VEGF. However, the mechanism that regulates the production of sVEGFR-1 by alternative polyadenylation remains poorly understood. In this review, we introduce and discuss the mechanism of alternative polyadenylation of VEGFR-1 mediated by protein arginine methylation.

UV-Pre-Treated and Protein-Adsorbed Titanium Implants Exhibit Enhanced Osteoconductivity.

Titanium materials are essential treatment modalities in the medical field and serve as a tissue engineering scaffold and coating material for medical devices. Thus, there is a significant demand to improve the bioactivity of titanium for therapeutic and experimental purposes. We showed that ultraviolet light (UV)-pre-treatment changed the protein-adsorption ability and subsequent osteoconductivity of titanium. Fibronectin (FN) adsorption on UV-treated titanium was 20% and 30% greater after 1-min and 1-h incubation, respectively, than that of control titanium. After 3-h incubation, FN adsorption on UV-treated titanium remained 30% higher than that on the control. Osteoblasts were cultured on titanium disks after 1-h FN adsorption with or without UV-pre-treatment and on titanium disks without FN adsorption. The number of attached osteoblasts during the early stage of culture was 80% greater on UV-treated and FN-adsorbed (UV/FN) titanium than on FN-adsorbed (FN) titanium; osteoblasts attachment on UV/FN titanium was 2.6- and 2.1-fold greater than that on control- and UV-treated titanium, respectively. The alkaline phosphatase activity of osteoblasts on UV/FN titanium was increased 1.8-, 1.8-, and 2.4-fold compared with that on FN-adsorbed, UV-treated, and control titanium, respectively. The UV/FN implants exhibited 25% and 150% greater in vivo biomechanical strength of bone integration than the FN- and control implants, respectively. Bone morphogenetic protein-2 (BMP-2) adsorption on UV-treated titanium was 4.5-fold greater than that on control titanium after 1-min incubation, resulting in a 4-fold increase in osteoblast attachment. Thus, UV-pre-treatment of titanium accelerated its protein adsorptivity and osteoconductivity, providing a novel strategy for enhancing its bioactivity.

Tuning of Titanium Microfiber Scaffold with UV-Photofunctionalization for Enhanced Osteoblast Affinity and Function.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3-10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2-15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.

Inhibition of Dipeptidyl Peptidase-4 Accelerates Epithelial-Mesenchymal Transition and Breast Cancer Metastasis via the CXCL12/CXCR4/mTOR Axis.

Dipeptidyl peptidase (DPP)-4 is a multifunctional glycoprotein involved in various biological and pathologic processes. DPP-4 has been widely recognized as a therapeutic target for type 2 diabetes mellitus but is also implicated in the development of human malignancies. Here, we show that inhibition of DPP-4 accelerates breast cancer metastasis via induction of CXCL12/CXCR4, which activates mTOR to promote epithelial-mesenchymal transition (EMT). In cultured cells, DPP-4 knockdown induced EMT and cell migration. Treatment with the DPP-4 inhibitor KR62436 (KR) promoted primary tumor growth and lung metastasis in a 4T1 tumor allograft mouse model; DPP-4 knockdown in 4T1 cells displayed similar phenotypes in vivo and in vitro. KR treatment enhanced the levels of CXCL12/CXCR4 and phosphorylated mTOR, which were associated with the induction of EMT in metastatic cancer cells. KR-induced EMT in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 or the mTOR inhibitor rapamycin, and AMD3100 suppressed KR-induced metastasis in vivo. Our findings suggest that DPP-4 plays a significant role in cancer biology and that inhibition of DPP-4 promotes cancer metastasis via induction of the CXCL12/CXCR4/mTOR/EMT axis. SIGNIFICANCE: These findings reveal that inhibition of DPP-4 increases the metastatic potential of breast cancer. This is especially important given the potential use of DPP-4 inhibition as a therapeutic strategy for type 2 diabetes.

Effects of Ultraviolet Photofunctionalization on Bone Augmentation and Integration Capabilities of Titanium Mesh and Implants.

PURPOSE: Ultraviolet (UV)-mediated photofunctionalization has earned considerable attention for the enhancement of the biologic capabilities of titanium. The effects of photofunctionalization on bone augmentation and gap closure were examined using titanium implants and mesh in a rat femur model. MATERIALS AND METHODS: An acid-etched titanium implant (4-mm length, 1-mm diameter) was placed in the gluteal tuberosity that resembles a knife-edge-like edentulous ridge. The lower half of the implant was located in a 2-mm-diameter defect created in the bone without cortical bone support; the upper half was exposed and covered with a titanium mesh to provide augmentation space. After 12 and 24 days of healing, specimens were subjected to microcomputed tomography (micro-CT)- and histology-based bone morphometry in three zones of analysis: augmentation, cortical bone-implant gap, and bone marrow. A biomechanical push-in test was performed to examine the strength of bone-implant integration. Photofunctionalization was performed by treating titanium implants and mesh with UV light for 12 minutes. RESULTS: Photofunctionalized titanium mesh and implants were hydrophilic, whereas untreated controls were hydrophobic. Bone volume was significantly greater in photofunctionalized implants and mesh than in untreated implants in all zones on days 12 and 24. Bone-to-implant contact of photofunctionalized implants was greater than that of untreated implants, not just in the bone marrow but also in the gap and augmented zones. The strength of osseointegration was three times greater for photofunctionalized implants than for untreated implants. CONCLUSION: Use of photofunctionalized titanium mesh and implants effectively enhanced vertical bone augmentation, cortical bone-implant gap closure, and osseointegration without innate bone support.

Comparison of MMP2 and MMP9 expression levels between primary and metastatic regions of oral squamous cell carcinoma.

Matrix metalloproteinases (MMPs) and tumor-associated macrophages (TAMs) play important roles in tumor growth. The present study investigated the expression levels of MMP2 and MMP9 in relation to the distribution of TAMs in the primary and metastatic regions of oral squamous cell carcinoma. Twenty-nine cases of oral squamous cell carcinoma (OSCC) with regional lymph node metastasis were selected from available documents in the archives of the Department of Pathology, Nihon University School of Dentistry. Four-micrometer-thick sections were prepared from the primary and metastatic regions. Each section was subjected to immunohistochemical staining using anti-MMP2, anti-MMP9, and anti-CD68 antibodies. The distribution and localization of MMPs and TAMs were compared between primary and metastatic regions. The expression levels of both MMPs were higher in the metastatic regions of lingual and gingival cancers. Statistically significant differences were observed in both T1 and T2 cases. In contrast to the higher expression of MMPs in metastatic regions, a higher number of TAMs were distributed in the primary regions. From these results, MMP expression levels and the numbers of TAMs were expected to have an inverse relationship between the primary and metastatic regions of OSCC. (J Oral Sci 58, 59-65, 2016).

Regulation of soluble Flt-1 (VEGFR-1) production by hnRNP D and protein arginine methylation.

Soluble fms-like tyrosine kinase-1 (sFlt-1) functions as a potent inhibitor of angiogenesis by trapping vascular endothelial growth factor (VEGF). However, the precise regulatory mechanism of sFlt-1 production is unknown. Here, we report that vascular sFlt-1 production is regulated by heterogeneous nuclear ribonucleoprotein D (hnRNP D) and arginine methylation. We showed that hnRNP D bound to Flt-1 pre-mRNA and that hnRNP D overexpression decreased sFlt-1 mRNA in human microvascular endothelial cells (HMVECs). In contrast, the reduction of hnRNP D levels induced an increase in sFlt-1 production. Overexpression of an hnRNP D mutant in which the arginine residue of the known arginine methylation motif (arginine-glycine-glycine; RGG) was replaced with alanine did not reduce the level of soluble-form RNA produced from the Flt-1 minigene. Moreover, we demonstrated that overexpression of arginine methyltransferase decreased the soluble-form RNA level, whereas overexpression of arginine demethylase and addition of methyltransferase inhibitors increased sFlt-1 mRNA levels. These findings indicate that hnRNP D and arginine methylation play important roles in the regulation of Flt-1 mRNA alternative polyadenylation.

Bone Generation Profiling Around Photofunctionalized Titanium Mesh.

PURPOSE: The aim of this study was to evaluate whether photofunctionalization of titanium mesh enhances its osteoconductive capability. MATERIALS AND METHODS: The titanium mesh (0.2 mm thickness) used in this study was made of commercially pure grade-2 titanium and had hexagonal apertures (2 mm width). Photofunctionalization was performed by treating titanium mesh with UV light for 12 minutes using a photo device immediately before use. Untreated or photofunctionalized titanium mesh was placed into rat femurs, and bone generation around titanium mesh was profiled using three-dimensional (3D) microcomputed tomography (micro-CT). A set of in vitro experiments was conducted using bone marrow-derived osteoblasts. RESULTS: Photofunctionalized titanium mesh surfaces were characterized by the regenerated hydrophilicity and significantly reduced surface carbon. Bone generation profiling at week 3 of healing showed that the hexagonal apertures in photofunctionalized mesh were 95% filled, but they were only 57% filled in untreated mesh, particularly with the center zone remaining as a gap. Bone profiling in slices parallel to the titanium surface showed that photofunctionalized titanium mesh achieved 90% bone occupancy 0 to 400 µm from the surface, compared with only 35% for untreated mesh. Bone occupancy remained as high as 55% 800 to 1,200 µm from photofunctionalized titanium mesh surfaces, compared with less than 20% for untreated mesh. In vitro, photofunctionalized titanium mesh expedited and enhanced attachment and spread of osteoblasts, and increased ALP activity and the rate of mineralization. CONCLUSION: This study may provide novel and advanced metrics describing the osteoconductive property of photofunctionalized titanium mesh. Specifically, photofunctionalization not only increased the breadth, but also the 3D range, of osteoconductivity of titanium mesh, enabling space-filling and far-reaching osteoconductivity. Further translational and clinical studies are warranted to establish photofunctionalized titanium mesh as a novel clinical tool for better bone regeneration and augmentation.

Usefulness of non-contrast-enhanced MRI with two-dimensional balanced steady-state free precession for the acquisition of the pulmonary venous and left atrial anatomy pre catheter ablation of atrial fibrillation: Comparison with contrast enhanced CT in clinical cases.

BACKGROUND: To investigate the feasibility of substituting non-contrast-enhanced MR (non-CE-MR) imaging with a two-dimensional (2D) balanced steady-state free precession (b-SSFP) sequence for contrast-enhanced computed tomography (CE-CT) for atrial fibrillation (AF) ablation. METHODS: Fifty-four patients that underwent AF ablation under the guidance of a 3D electro-anatomical mapping system with CE-CT (n = 27) or non-CE-MR images (n = 27) were studied. Procedural results were compared between the two groups. Furthermore, in 22 patients who underwent both CE-CT and non-CE-MRI, two cardiologists independently scored the multiplanar reformatted images on a scale of 1 to 4 (from 1, poor, to 4, excellent). RESULTS: The image score was nearly 0.5 point higher with the CE-CT method. However, the procedural results such as the surface registration error (1.0 [0.8-1.6] mm versus 1.0 [0.8-1.35] mm, P = 0.88) and procedure time (185 [159-199] min versus 185 [142-221] min, P = 0.86) did not significantly differ between the CE-CT and non-CE-MR groups. CONCLUSION: The non-CE-MR method with a 2D-b-SSFP sequence can give us adequate information on AF ablation without any radiation exposure or contrast medium usage

Effect of UV Photofunctionalization on Biologic and Anchoring Capability of Orthodontic Miniscrews.

PURPOSE: Treatment of titanium with UV light immediately before use, or photofunctionalization, is gaining traction as a simple method to improve the biologic capability and clinical performance of dental implants. The objective of this study was to examine the effect of photofunctionalization on the biologic capability and mechanical anchorage of orthodontic miniscrews. MATERIALS AND METHODS: Untreated and photofunctionalized Ti-6Al-4V orthodontic miniscrews were placed into rat femurs. Photofunctionalization was performed by treating miniscrews with UV light for 12 minutes using a photo device immediately before placement. After 3 weeks of healing, miniscrews were pushed laterally to measure the resistance against the tipping force. The miniscrews were also evaluated for morphology and chemistry of tissue formed around them using scanning electron microscopy and energy dispersive spectroscopy. Rat bone marrow-derived osteoblasts were cultured on Ti-6Al-4V disks with and without photofunctionalization. The number of osteoblasts attached to the disks and the behaviors, alkaline phosphatase activity, and mineralization capability of osteoblasts were evaluated. RESULTS: Photofunctionalization converted both disk and screw surfaces from hydrophobic to superhydrophilic. In vivo biomechanical testing showed that the displacement of untreated screws was 1.5 to 1.7 times greater than that of photofunctionalized screws when subjected to lateral tipping force. Robust bone formation was observed around photofunctionalized miniscrews with strong elemental peaks of calcium and phosphorus, whereas the tissue around untreated miniscrews appeared thin and showed no clear peak of calcium. The attachment, initial spreading, adhesion, and expression of functional phenotypes of osteoblasts were significantly increased on photofunctionalized Ti-6Al-4V disks. CONCLUSION: These in vivo and in vitro results comprehensively and consistently demonstrate that photofunctionalization increases the bioactivity of Ti-6Al-4V and improves the anchoring capability of orthodontic miniscrews.

Ultraviolet photofunctionalization increases removal torque values and horizontal stability of orthodontic miniscrews.

INTRODUCTION: The objective of this study was to examine the effects of ultraviolet-mediated photofunctionalization of miniscrews and the in-vivo potential of bone-miniscrew integration. METHODS: Self-drilling orthodontic miniscrews made from a titanium alloy were placed in rat femurs. Photofunctionalization was performed by treating the miniscrews with ultraviolet light for 12 minutes with a photo device immediately before implantation. Maximum insertion torque (week 0), removal torque (weeks 0 and 3), and resistance to lateral tipping force (week 3) were examined. RESULTS: The removal torque at 3 weeks of healing was higher for the photofunctionalized screws than for the untreated screws. The regenerated bone tissue was more intact and contiguous around the photofunctionalized miniscrews than around the untreated ones. The miniscrew-bone complex seemed to produce interface failure, not cohesive fracture, in both groups. The displacement of untreated screws under a lateral tipping force was greater than that of photofunctionalized miniscrews. CONCLUSIONS: These results suggest that photofunctionalization increases the bioactivity of titanium-alloy miniscrews and improves the anchoring capability of orthodontic miniscrews, even without modification of the surface topography.

Melanocortins contribute to sequential differentiation and enucleation of human erythroblasts via melanocortin receptors 1, 2 and 5.

In this study, we showed that adrenocorticotropic hormone (ACTH) promoted erythroblast differentiation and increased the enucleation ratio of erythroblasts. Because ACTH was contained in hematopoietic medium as contamination, the ratio decreased by the addition of anti-ACTH antibody (Ab). Addition of neutralizing Abs (nAbs) for melanocortin receptors (MCRs) caused erythroblast accumulation at specific stages, i.e., the addition of anti-MC2R nAb led to erythroblast accumulation at the basophilic stage (baso-E), the addition of anti-MC1R nAb caused accumulation at the polychromatic stage (poly-E), and the addition of anti-MC5R nAb caused accumulation at the orthochromatic stage (ortho-E). During erythroblast differentiation, ERK, STAT5, and AKT were consecutively phosphorylated by erythropoietin (EPO). ERK, STAT5, and AKT phosphorylation was inhibited by blocking MC2R, MC1R, and MC5R, respectively. Finally, the phosphorylation of myosin light chain 2, which is essential for the formation of contractile actomyosin rings, was inhibited by anti-MC5R nAb. Taken together, our study suggests that MC2R and MC1R signals are consecutively required for the regulation of EPO signal transduction in erythroblast differentiation, and that MC5R signal transduction is required to induce enucleation. Thus, melanocortin induces proliferation and differentiation at baso-E, and polarization and formation of an actomyosin contractile ring at ortho-E are required for enucleation.

Effect of ultraviolet-mediated photofunctionalization for bone formation around medical titanium mesh.

PURPOSE: The new technology of photofunctionalization with ultraviolet (UV) light for titanium implants has earned considerable attention. We hypothesized that UV light treatment would enhance bone formation on titanium mesh. MATERIALS AND METHODS: We implemented in vitro and in vivo experiments to examine the effectiveness of UV treatment for bone formation on titanium mesh surfaces. Titanium mesh for medical use was prepared as samples, which were autoclaved and stored under dark ambient conditions for 4 weeks. UV treatment was performed for 12 minutes. Carbon contamination, hydrophilicity, and protein adhesion of the titanium mesh surface were examined in an in vitro model. Bone tissue formation around the titanium mesh was observed in a rat femur bone model. The Mann-Whitney U test was used to examine differences between the untreated and UV-treated groups. P values of < .05 were considered significant. RESULTS: UV-mediated photofunctionalization reduced carbon contamination rates on the untreated titanium mesh surfaces. The hydrophobic surface of the untreated titanium mesh became superhydrophilic after UV-mediated photofunctionalization (P < .01). The amount of protein adsorbed onto the titanium was 1.5 to 3 times greater on the photofunctionalized titanium mesh surfaces than on the untreated titanium mesh surfaces (P < .01). In the animal experiment, the newly formed bone on the UV-treated titanium mesh was approximately 2.5 times greater than that on the untreated mesh (P < .05). CONCLUSIONS: UV-mediated photofunctionalization is effective, as demonstrated by the enhanced bone tissue formation on the titanium mesh. Future studies will focus on bone augmentation using an UV-mediated photofunctionalized titanium implant and mesh.

N-acetyl cysteine as an osteogenesis-enhancing molecule for bone regeneration.

Bone regeneration often requires cues from osteogenesis-inducing factors for successful outcome. N-acetyl cysteine (NAC), an anti-oxidant small molecule, possibly modulates osteoblastic differentiation. This study investigated the potential of NAC as an osteogenesis-enhancing molecule in vitro and in vivo. Various concentrations of NAC (0, 2.5, 5.0, and 10 mM) were added to rat bone marrow stromal cell or osteoblastic cell culture in media with or without dexamethasone. The results showed marked enhancement of alkaline phosphatase activity and mineralized matrix formation together with consistent upregulation of bone-related gene markers such as collagen I, osteopontin, and osteocalcin in the osteoblastic culture with addition of 2.5 or 5.0 mM NAC regardless of the presence of dexamethasone. Micro-CT-based analysis and histological observation revealed that addition of NAC to a collagenous sponge implanted in a critical size cortical bone defect (3.0 mm × 5.0 mm) in rat femur yielded acceleration and completion of defect closure, with thick, compact, and contiguous bone after 6 weeks of healing. In contrast, with sponge alone, only sparse and incomplete bone regeneration was observed during the matching healing period. These results indicate that NAC can function as an osteogenesis-enhancing molecule to accelerate bone regeneration by activating differentiation of osteogenic lineages.