Investigation of a new implant surface modification using phosphorylated pullulan.

Various implant surface treatment methods have been developed to achieve good osseointegration in implant treatment. However, some cases remain impossible to treat with implants because osseointegration is not obtained after implantation, and the implants fail. Thus, this study focused on phosphorylated pullulan because of its adhesiveness to titanium (Ti) and bone, high biocompatibility, and early replacement with bone. In this study, the response of bone-related cells to phosphorylated pullulan was evaluated to develop a new surface treatment method. Saos-2 (human osteosarcoma-derived osteoblast-like cells), MC3T3-E1 (mouse calvaria-derived osteoblast-like cells), and RAW264.7 (mouse macrophage-like cells) were used. In evaluating cellular responses, phosphorylated pullulan was added to the culture medium, and cell proliferation and calcification induction tests were performed. The proliferation and calcification of cells on the surface of Ti disks coated with phosphorylated pullulan were also evaluated. In addition, bone morphogenetic protein-2 (BMP-2), an osteogenic factor, was used to evaluate the role of phosphorylated pullulan as a drug carrier in inducing calcification on Ti disks. Phosphorylated pullulan tended to promote the proliferation of osteoblast-like cells and the formation of calcification on Ti disks coated with phosphorylated pullulan. Ti disks coated with phosphorylated pullulan loaded with BMP-2 enhanced calcification. Phosphorylated pullulan inhibited osteoclast-like cell formation. These results are due to the properties of phosphorylated pullulan, such as adhesiveness to titanium and drug-loading function. Therefore, phosphorylated pullulan effectively promotes bone regeneration when coated on titanium implants and is useful for developing a new surface treatment method.

Pharmacological control of angiogenesis by regulating phosphorylation of myosin light chain 2.

BACKGROUND: Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis. METHODS AND RESULTS: We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs. CONCLUSION: The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis.

Extended and adjustable field-of-view of variable interscan time analysis by ammonite-scanning swept-source optical coherence tomography angiography.

A novel scanning protocol, ammonite scan, is proposed for widefield optical coherence tomography angiography (OCTA) and relative retinal blood flow velocity imaging in the human retina using variable interscan time analysis (VISTA). A repeated circle scan using a 400 kHz swept-source was employed to achieve an interscan time of 1.28 ms. The center of the repeated circular scan continuously moved spirally towards the peripheral region, ensuring an extended and adjustable scan range while preserving the short interscan time. Image artifacts due to eye movement were eliminated via extra motion-correction processing using data redundancy. The relative blood flow velocity in superficial and deep plexus layers was calculated from the VISTA image, and their ratio was used to explore the microvascular flow parameter in the healthy human eye.

Application of a Titanium Screw for the Hemorrhage from the Bone Channel during the Lateral Window Technique: A Technical Note.

Pulsatile and profuse hemorrhage occurred in the lateral window technique for implant placement. The surgery was performed in the dental clinic under local anesthesia. The posterior superior alveolar artery was suspected to be the main feeder. Ordinary methods for hemostasis, such as vasoconstrictor-soaked gauze compression, electrocautery hemostasis, absorbable hemostat packing, and bone wax application, were tried. However, strong pulsatile bleeding could not be controlled at all. Complete hemostasis was hardly expected. The idea came up when the titanium screws came into sight. The sterilized screw was always stocked for bone grafting. After visualizing the bleeding point clearly by suction, and the screw was inserted into the bone channel. The bleeding was completely stopped immediately. It may not be a novel method, but is certainly a reliable application of the screw, which is fundamentally the same as arterial catheter embolization.

Automated cell isolation from photodegradable hydrogel based on fluorescence image analysis.

We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO2 concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.

Fibroepithelial urethral polyp with minor prostatic glands in an older man.

We describe a man in his 60s with an incidentally detected urethral polyp located in the middle section of his penile shaft. The patient had been suffering from urinary retention for 9 years due to benign prostatic hyperplasia. He was initiated on clean intermittent catheterisation to drain urine 1 year before the detection of the urethral polyp. Holmium laser nucleation of the prostate was performed, and an anterior urethral polyp (5 mm in diameter) was resected. Pathology indicated that the urethral polyp was a fibroepithelial polyp with prostate-specific antigen-immunoreactive heterotopic prostatic glands. There were no problems with urination 6 months after surgery. Fibroepithelial urethral polyps are usually congenital and are rarely described in adults. The clinicopathological features of this polyp, with its uniquely associated minor prostatic glands, are presented.

A Novel Variation of the Lateral Window Approach for Sinus Floor Elevation in a Case of Bony Nodular Prominence.

Introduction: In sinus floor augmentation, bony nodular prominence at the floor of the maxillary sinus is an obstruction to lateral window approach. It is challenging to detach and elevate the sinus membrane without making any perforations around the nodular prominence, because the membrane is very thin. To overcome these difficulties, we developed a novel method. Method: The membrane was not detached from the surface of the nodular prominence except for at the basal point, but the nodular prominence was cut at the base. Nodular prominence and sinus membrane were elevated upward together. A resorbable collagen membrane was placed beneath it to provide mechanical support and cover any partial tear in the sinus membrane. Autogenous bone and/or bone substitute were packed in the vacant compartment depending on the condition. Conclusion: This method is very easy to be carried out without any difficulties and worries. It gives us great benefit on the sinus floor augmentation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12663-021-01572-7.

Basic fibroblast growth factor uniquely stimulates quiescent vascular smooth muscle cells and induces proliferation and dedifferentiation.

Blood vessels normally remain stable over the long term. However, in atherosclerosis, vascular cells leave the quiescent state and enter an activated state. Here, we investigated the factors that trigger the breakage of the quiescent state by screening growth factors and cytokines using a vascular smooth muscle cell (SMC) line and an endothelial cell (EC) line. Despite known functions of the tested factors, only basic fibroblast growth factor (bFGF) was identified as a potent trigger of quiescence breakage in SMCs, but not ECs. bFGF disrupted tight SMC-monolayers and caused morphological changes, proliferation, and dedifferentiation. Human primary SMCs, but not ECs, also showed similar results. Aberrant SMC proliferation is a critical histological event in atherosclerosis. We, thus, provide further insights into the role of bFGF in vascular pathobiology.

Iron Deficiency Anemia Improved by Dental Implantation: A Case Report.

Treatments for improving iron deficiency anemia are generally aimed at increasing oral iron intake and/or administration. Such treatments, however, have been unsuccessful in managing nutritional disorders, including anemia, in patients with masticatory dysfunction caused by impaired occlusion. Nevertheless, few studies have assessed the potential benefits of providing optimal occlusion in such cases. Here, we report a case involving a 53-year-old woman with iron deficiency anemia, wherein we attempted to facilitate efficient mastication by establishing functional occlusion with dental implant placement. The patient was diagnosed with iron deficiency anemia and hospitalized for blood transfusion 2 years before she visited our dental clinic. At the first visit, her hemoglobin (Hb) and mean corpuscular volume values were low; sodium ferrous citrate administration and dietary guidance led to slight improvement. However, blood transfusions and iron supplementation had been ineffective over longer duration. After dental implant placement, her Hb and mean corpuscular volume values were restored and maintained for >4 years without medication. Through this report, we highlight an alternative, non-pharmacological treatment strategy for iron deficiency anemia.

Adrenal Schwannoma in an Elderly Man: A Case Report and Literature Review.

Schwannoma is a common mesenchymal neoplasm; however, adrenal schwannoma is rare, and it is frequently misdiagnosed as adrenal cortical adenoma. We herein report a 91-year-old Japanese man with right adrenal schwannoma that was pathologically diagnosed after adrenalectomy. To our knowledge, this is the first case of adrenal schwannoma in the oldest patient and with the longest follow-up period reported, including radiological images from 10 years earlier.

Sensorless astigmatism correction using a variable cross-cylinder for high lateral resolution optical coherence tomography in a human retina.

High lateral resolution (∼5µm) optical coherence tomography (OCT) that employs a variable cross-cylinder (VCC) to compensate for astigmatism is presented for visualizing minute structures of the human retina. The VCC and its sensorless optimization process enable ocular astigmatism correction of up to -5.0 diopter within a few seconds. VCC correction has been proven to increase the signal-to-noise ratio and lateral resolution using a model eye. This process is also validated using the human eye by visualizing the capillary network and human cone mosaic. The proposed method is applicable to existing OCT, making high lateral resolution OCT practical in clinical settings.

Osteocytes as main responders to low-intensity pulsed ultrasound treatment during fracture healing.

Ultrasound stimulation is a type of mechanical stress, and low-intensity pulsed ultrasound (LIPUS) devices have been used clinically to promote fracture healing. However, it remains unclear which skeletal cells, in particular osteocytes or osteoblasts, primarily respond to LIPUS stimulation and how they contribute to fracture healing. To examine this, we utilized medaka, whose bone lacks osteocytes, and zebrafish, whose bone has osteocytes, as in vivo models. Fracture healing was accelerated by ultrasound stimulation in zebrafish, but not in medaka. To examine the molecular events induced by LIPUS stimulation in osteocytes, we performed RNA sequencing of a murine osteocytic cell line exposed to LIPUS. 179 genes reacted to LIPUS stimulation, and functional cluster analysis identified among them several molecular signatures related to immunity, secretion, and transcription. Notably, most of the isolated transcription-related genes were also modulated by LIPUS in vivo in zebrafish. However, expression levels of early growth response protein 1 and 2 (Egr1, 2), JunB, forkhead box Q1 (FoxQ1), and nuclear factor of activated T cells c1 (NFATc1) were not altered by LIPUS in medaka, suggesting that these genes are key transcriptional regulators of LIPUS-dependent fracture healing via osteocytes. We therefore show that bone-embedded osteocytes are necessary for LIPUS-induced promotion of fracture healing via transcriptional control of target genes, which presumably activates neighboring cells involved in fracture healing processes.

Stepwise construction of dynamic microscale concentration gradients around hydrogel-encapsulated cells in a microfluidic perfusion culture device.

Inside living organisms, concentration gradients dynamically change over time as biological processes progress. Therefore, methods to construct dynamic microscale concentration gradients in a spatially controlled manner are needed to provide more realistic research environments. Here, we report a novel method for the construction of dynamic microscale concentration gradients in a stepwise manner around cells in micropatterned hydrogel. In our method, cells are encapsulated in a photodegradable hydrogel formed inside a microfluidic perfusion culture device, and perfusion microchannels are then fabricated in the hydrogel by micropatterned photodegradation. The cells in the micropatterned hydrogel can then be cultured by perfusing culture medium through the fabricated microchannels. By using this method, we demonstrate the simultaneous construction of two dynamic concentration gradients, which allowed us to expose the cells encapsulated in the hydrogel to a dynamic microenvironment.

The role of PI3K/Akt/mTOR signaling in dose-dependent biphasic effects of glycine on vascular development.

Glycine, a non-essential amino acid, exerts concentration-dependent biphasic effects on angiogenesis. Low-doses of glycine promote angiogenesis, whereas high-doses cause anti-angiogenesis. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling participates in angiogenesis of both physiological development, and pathological events including tumor and inflammation. We assessed the role of PI3K/Akt/mTOR signaling in vascular development, and the interaction with glycine, using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos expressing fluorescent proteins in vascular endothelial cells. Treatment with inhibitors of mTORC1 (rapamycin and everolimus), mTORC1/mTORC2 (KU0063794), PI3K (LY29400), and Akt (Akt inhibitor) decreased the development of intersegmental vessels (ISVs). These inhibitors cancelled the angiogenic effects of a low-dose of glycine, while acted synergistically with a high-dose of glycine in anti-angiogenesis. mTOR signaling regulates the gene expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and nitric oxide (NO) synthase (NOS), an enzyme for the synthesis of an angiogenic mediator NO. Expressions of VEGF and NOS were consistent with the vascular features induced by glycine and an mTOR inhibitor. Our results suggest that PI3K/Akt/mTOR signaling may interact with dose-dependent biphasic effects of exogenous glycine on in vivo angiogenesis. mTOR signaling is a key target for cancer therapy, thus, the combining mTOR inhibitors with glycine may be a potential approach for controlling angiogenesis.

Fabrication of Hollow Structures in Photodegradable Hydrogels Using a Multi-Photon Excitation Process for Blood Vessel Tissue Engineering.

Engineered blood vessels generally recapitulate vascular function in vitro and can be utilized in drug discovery as a novel microphysiological system. Recently, various methods to fabricate vascular models in hydrogels have been reported to study the blood vessel functions in vitro; however, in general, it is difficult to fabricate hollow structures with a designed size and structure with a tens of micrometers scale for blood vessel tissue engineering. This study reports a method to fabricate the hollow structures in photodegradable hydrogels prepared in a microfluidic device. An infrared femtosecond pulsed laser, employed to induce photodegradation via multi-photon excitation, was scanned in the hydrogel in a program-controlled manner for fabricating the designed hollow structures. The photodegradable hydrogel was prepared by a crosslinking reaction between an azide-modified gelatin solution and a dibenzocyclooctyl-terminated photocleavable tetra-arm polyethylene glycol crosslinker solution. After assessing the composition of the photodegradable hydrogel in terms of swelling and cell adhesion, the hydrogel prepared in the microfluidic device was processed by laser scanning to fabricate linear and branched hollow structures present in it. We introduced a microsphere suspension into the fabricated structure in photodegradable hydrogels, and confirmed the fabrication of perfusable hollow structures of designed patterns via the multi-photon excitation process.

Glycine exerts dose-dependent biphasic effects on vascular development of zebrafish embryos.

Glycine, a non-essential amino acid, is involved in both angiogenesis and anti-angiogenesis. We hypothesized that glycine would exert dose-dependent different effects on angiogenesis. In this study, we investigated the effects of a broad range of concentrations of glycine on vascular development using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos. Effects of glycine transporter (GlyT) inhibitors (sarcosine and bitopertin) and a glycine receptor (GlyR) inhibitor (strychnine) were also examined in embryos in the absence or presence of glycine. After exposure to glycine and inhibitors, intersegmental vessels (ISVs) were observed by fluorescent microscopy. Low concentrations of glycine promoted the development of ISVs, whereas high concentrations reduced it. These effects of glycine could generally be reversed by treatment with GlyT and GlyR inhibitors. Furthermore, expressions of vascular endothelial growth factor (VEGF) (an angiogenic factor) and nitric oxide synthase (NOS) (an enzyme for nitric oxide synthesis) were associated with the dose-dependent effects of glycine. Our results suggest that glycine exerts dose-dependent biphasic effects on vascular development, which rely on GlyTs and GlyRs, and correlate with the expression of VEGF and NOS genes. At low concentrations, glycine acted as an angiogenic factor. In contrast, at high concentrations, glycine induced anti-angiogenesis. This evidence provides a novel insight into glycine as a unique target in angiogenic and anti-angiogenic therapy.

Histone H3K9 methylation is involved in temporomandibular joint osteoarthritis.

The morbidity of temporomandibular joint osteoarthritis (TMJOA) increases with age. Condylar articular cartilage degradation, which causes TMJOA, is known to be involved in articular chondrocyte metabolic imbalances in the temporomandibular joint (TMJ) and in other joints of the body. Epigenetic regulation, such as the chemical modification of DNA and histones, is implicated in cartilage homeostasis. However, few studies have been conducted on the epigenetic regulation of condylar articular cartilage degradation. The present study investigated the regulation of histone H3 lysine 9 (H3K9) methylation and its effects on the pathogenesis of degenerative TMJ cartilage disorders. The histone H3K9 methylation level was decreased in degenerated condylar articular cartilage in aged mice. Treatment with chaetocin (a selective H3K9 methylation inhibitor) reduced cell viability and promoted caspase­3/7 activity in ATDC5 mouse chondroprogenitor cells. The inhibition of H3K9 methylation increased matrix metalloproteinase (Mmp)1 and Mmp13 mRNA expression in these cells. Furthermore, the expression levels of Sox9 and collagen α1(II) (Col2a1) mRNA, which are anabolic factors for chondrogenic differentiation, were also decreased by treatment with chaetocin, which is an inhibitor of histone methyltransferases. These results indicated that histone H3K9 methylation regulates chondrocyte homeostasis in terms of cell growth, apoptosis and gene expression, and highlighted a possible future therapy option for TMJOA.

Osteoadherin serves roles in the regulation of apoptosis and growth in MC3T3­E1 osteoblast cells.

Small leucine­rich proteoglycans (SLRPs) are a class of proteoglycans that are characterized by small protein cores and structures of leucine­rich repeats. SLRPs are expressed in most extracellular matrices and share numerous biological functions that are associated with binding of collagens and cell surface receptors. Osteoadherin (also termed osteomodulin) is encoded by the Omd gene and is a keratan sulfate proteoglycan of the class II subfamily of SLRPs. Osteoadherin is highly expressed in mineralized tissues, including bone and dentin; however, it's precise roles remain unknown. The present study determined the Omd expression levels and investigated the effects of over­ and under­expression of osteoadherin in osteoblastic cells. Omd mRNA expression increased with osteoblast differentiation in MC3T3­E1 cells. In C2C12 cells, Omd mRNA expression was induced by bone morphogenetic protein (BMP)2. Reporter assays similarly demonstrated activation of the Omd gene promoter following co­transfection with Smad1 and Smad4, which are intracellular signaling molecules of the BMP2 signaling pathway. Overexpression of Omd increased the viability and decreased caspase 3/7 activity in MC3T3­E1 cells. By contrast, following transfection with small interfering RNA for Omd, viable cell numbers were decreased and caspase 3/7 activity was increased. Furthermore, overexpression of Omd reduced the expression of CCN family 2 in these cells. These results demonstrate that Omd expression is regulated during osteoblast differentiation, and that the protein product osteoadherin serves roles in the apoptosis and growth of osteoblast cells.

Can the Partial Peptide SIVSF of the ß2-Adrenergic Receptor Recognize Chirality of the Epinephrine Neurotransmitter?

Chirality plays an essential role in biological molecular recognition, such as neurotransmission. Here, we applied electrospray-cold ion trap spectroscopy to complexes of a partial binding motif SIVSF of a ß2-adrenergic receptor pocket with L- and D-epinephrine AdH+. The ultraviolet spectrum of the SIVSF-AdH+ complex changed drastically when L-AdH+ was replaced by its enantiomer. The isomer-selected infrared spectra revealed that D-AdH+ was bound to SIVSF by its protonated amino-group or a single catechol OH and induced nonhelical secondary structures of SIVSF. This is in sharp contrast to the helical SIVSF complex with L-AdH+, which is close to the natural binding structure with two catechol OHs binding in the receptor. This shows that a short pentapeptide SIVSF can distinguish the chirality of the ligand AdH+ as well as the receptor. This stereoselectivity is suggested to arise from an additional interaction involving the hydroxyl group on the chiral carbon.