Trabecular Cells Research Articles

Human stem cells home to and repair laser-damaged trabecular meshwork in a mouse model

Glaucoma is the leading cause of irreversible vision loss, and reducing elevated intraocular pressure is currently the only effective clinical treatment. The trabecular meshwork is the main resistance site for aqueous outflow that maintains intraocular pressure. In this study, we transplanted human trabecular meshwork stem cells (TMSCs) intracamerally into mice that received laser photocoagulation over a 180° arc of the trabecular meshwork. TMSCs preferentially homed and integrated to the laser-damaged trabecular meshwork region and expressed differentiated cell markers at 2 and 4 weeks. Laser-induced inflammatory and fibrotic responses were prevented by TMSC transplantation with simultaneous ultrastructure and function restoration. Cell affinity and migration assays and elevated expression of CXCR4 and SDF1 in laser-treated mouse trabecular meshwork suggest that the CXCR4/SDF1 chemokine axis plays an important role in TMSC homing. Our results suggest that TMSCs may be a viable candidate for trabecular meshwork refunctionalization as a novel treatment for glaucoma.

Differentiation of microfluidic-encapsulated trabecular meshwork mesenchymal stem cells into insulin producing cells and their impact on diabetic rats.

Tissue and stem cell encapsulation andtransplantation were considered as promising tools in the treatment of patients with diabetes mellitus. The aim of this study was to evaluate the effect of microfluidic encapsulation on the differentiation of trabecular meshwork mesenchymal stem cells (TM-MSC), into insulin-producing cells (IPCs) both in vitro and in vivo. The presence of differentiated cells in microfibers (three dimensional [3D]) and tissue culture plates (TCPS; two dimensional [2D]) culture was evaluated by detecting mRNA and protein expression of pancreatic islet-specific markers as well as measuring insulin release of cells in response to glucose challenges. Finally, semi-differentiated cells in microfibers (3D) and 2D cultures were used to control the glucose level in diabetic rats. The results of this study showed that MSCs differentiated in alginate microfibers (fabricated by microfluidic device) express more Pdx-1 mRNA (1.938-fold, p-value: 0.0425) and Insulin mRNA (2.841-fold, p-value: 0.0001) compared with those cultured on TCPS. Furthermore, cell encapsulation in microfluidic derived microfibers decreased the level of blood glucose in diabetic rats. The approach used in this study showed the possibility of alginate microfibers as a matrix for differentiation of TM-MSCs (as a new source) into IPCs. In addition, it could minimize different steps in stem cell differentiation, handling, and encapsulation, which lead to loss of an unlimited number of cells.

Hepatic Metastasis Presenting as Merkel Cell Carcinoma of Unknown Primary

Merkel cell carcinoma (MCC), a high grade cutaneous neuroendocrine tumor (NET), is often confused with basal cell carcinoma (BCC), basaloid squamous cell carcinoma (SCC), and other primary site small cell carcinomas. Usually limited to skin and regional lymph nodes, solid organ metastasis is uncommon and results in high mortality. We present a case of a man with abdominal pain and liver lesions who was diagnosed with Merkel cell carcinoma of unknown primary (MCCUP). A 74-year-old man presented with a one-week history of shortness of breath, pleuritic chest pain, and abdominal pain. Significant medical history included prostate adenocarcinoma and Moh's surgery for SCC and BCC. On examination, he demonstrated epigastric tenderness. Laboratory studies were remarkable for elevated liver enzymes and total bilirubin. Initial workup ruled out acute coronary syndrome and pulmonary embolism; however, chest CT showed multiple pulmonary nodules and a large peri-fissural nodule. Abdominal CT showed a mottled liver and lytic lesions in the vertebral column concerning for metastasis. A liver biopsy showed high-grade NET. Immunohistochemical (IHC) staining favoring MCC and excluding primary pulmonary and gastrointestinal SCC. Hospital course was complicated by worsening jaundice and ascites for which he underwent palliative chemotherapy without improvement. Repeat imaging showed worsening hepatic metastases as well as new lytic lesions. There was a decrease in size of the peri-fissural nodule, but no change in the other lung nodules. Palliative radiation therapy was elected and the patient passed away 4 months after presentation. MCC is a rare, aggressive NET, most common in Caucasians over age 65 with UV exposure. Most patients present with cutaneous disease and regional lymph node involvement. Systemic spread is rare; however, common metastatic sites include lymph nodes, lung, liver, and bone. MCCUP typically presents with nodal disease without known primary cutaneous MCC. Histologic diagnosis of MCCUP is difficult due to similarities with more common high-grade NETs, such as pulmonary or gastrointestinal small cell carcinoma. Key electron microscopic features include dense core granules and paranuclear intermediate filaments. CK-20 positivity, and TTF-1 and CDX-2 negativity on IHC staining, to exclude pulmonary and gastrointestinal SCC, are confirmatory. Outcomes for patients with MCC is poor; treatment typically involves palliative chemotherapy.2349_A Figure 1. Computed Tomography of the Abdomen w/contrast - The liver is enlarged with a heterogeneous and mottled appearance - Axial section2349_B Figure 2. Needle biopsy of liver showing a trabecular small cell carcinoma (MCC) at 200 magnification (H&E 20X)2349_C Figure 3. Merkel cell carcinoma marking with CK20 in a para-nuclear pattern at 600 magnification (60X)

Mechanisms of Glaucoma in Exfoliation Syndrome.

The most important characteristic of exfoliation syndrome (XFS) is that it involves a greater risk of developing glaucoma. In comparison with other forms of open-angle glaucoma, exfoliation glaucoma is more resistant to medical therapy and progresses faster. Possible pathologic mechanisms of glaucoma development in XFS comprise: (1) elevated intraocular pressure (IOP) caused by functional impairment of aqueous humor outflow due to deposition of exfoliation material in the trabecular meshwork and trabecular cell dysfunction, (2) XFS-associated connective tissue elastosis leading to structural and functional alterations of the lamina cribrosa which increases the vulnerability toward elevated IOP and development of glaucomatous optic neuropathy, (3) elevated IOP due to closure of the anterior chamber angle accompanied by forward displacement of the crystalline lens due to zonular weakness, (4) presumable primary functional impairment of retinal ganglion cells. In addition to the discovery of lysyl oxidase-like 1, further genetic associations have been identified and knowledge related to XFS etiology and pathophysiology has markedly increased over the past 10 years. Further cell biological investigations navigated by the molecular genetics underlying XFS will eventually lead to a better understanding of the complex mechanisms of exfoliation glaucoma.

F94. Predicted stimulus thresholds of thoracic pedicle screws in the pediatric population

Introduction Previous studies conducted on pedicle screws in lumbar spine surgeries have determined that electrophysiological monitoring using stimulus evoked electromyography (EMG) can reduce the risk of nerve root trauma based on specific stimulation threshold criteria. However, the validity and efficacy of traditional pedicle screw testing (PST) techniques applied to thoracic pedicle screws has been questioned. Furthermore, there is limited data regarding PST in the pediatric population. Data from pediatric scoliosis operations performed at C.S. Mott Children’s Hospital allows for a comprehensive analysis of thoracic pedicle screw stimulation values and corresponding post-operative status. Methods A retrospective review of thoracic PST in the pediatric population was performed. 167 pediatric orthopedic spinal corrective procedures utilizing thoracic pedicle screw implantation and electrical testing techniques were performed at C.S. Mott Children’s Hospital between June 2010 and August 2015. 1447 screws placed at the T8 - L1 levels were electrically tested using cathodal, monopolar stimulation with a pulse width of 200 μs and current intensity up to 20 mA. Alarm criteria thresholds were set to an observable compound muscle action potential (CMAP) from a corresponding muscle (rectus abdominus or iliopsoas) in response to a stimulating current of 6 mA or less. A retrospective review of stimulus thresholds, specific to each level and side was performed. For each spinal level the average stimulation threshold was calculated. Results Stimulation of 22 screws in 19 patients evoked a CMAP using current levels less than 2 standard deviations below the average stimulation threshold for that vertebral level. Eight of these screws tested above the established alert criteria of 6 mA. Of the remaining screws, 3 remained in place, 3 were removed and replaced, and 7 were removed and left out. None of the 19 patients had post-operative deficits. Conclusion These findings are inconclusive regarding the efficacy of current thoracic pedicle screw testing methods. It is possible that PST at these spinal levels is unreliable due to the wide variability in bone density and cortical and trabecular bone cell maturity in pediatric patients. It is also possible there were electrophysiological breaches of the pedicle wall though these were not clinically significant due to a safe margin between the pedicle screw and nerve root. ( Prospective ) Longitudinal studies should be conducted to provide data on the long-term outcomes of these patients. Additionally, future research should explore the use of multi-pulse stimulation in pediatrics as well as the sensitivity of EMG responses in the abdominals and iliopsoas to detect nerve root trauma.

Expression Profiles of eNOS, iNOS, endothelin receptors and microRNAs‐27b, 155 and 199 in the corpus cavernosum of rats submitted to chronic alcoholism and diabetes mellitus.

IntroductionEthanol consumption has been described to increase the levels of plasma endothelin‐1 (ET‐1) suggesting that chronic consumption activates endothelinergic pathway and also to affect nitric oxide synthase (NOS) derived nitric oxide (NO) and NOS expression in different tissues. Recent evidence showed that chronic ethanol consumption increases ET‐1‐induced sustained contraction of trabecular smooth muscles cells of the corpora cavernosal in corpus cavernosum of rat by mechanism that involves increased expression of ETA eee and ETB receptors.ObjectivesTo evaluate the effects of chronic ethanol consumption associated with diabetes affected the endothelin system in the cavernosal smooth muscle (CSM) of rats, as well as the effects of chronic ethanol consumption and diabetes on nitric oxide (NO)‐mediated relaxation of cavernosal smooth muscle (CSM).Materials and MethodsCorpus cavernosum were divided into four groups: control, ethanol, diabetic and ethanol‐diabetic of Male Wistar rats. We analyzed protein and gene expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and endothelin receptors (ETA eeeand ETB) by immunohistochemistry and real time‐PCR. The expression of miRNAs‐27a, 199 and 155 by real time‐PCR in corpus cavernosum. It was also evaluated the effect of ethanol consumption associated with diabetes on the relaxation induced by acetylcholine (ACh; 0.01–1000 micromol/L) and in the contraction induced by Endothelin‐1 (ET‐1 0.1 nmol‐ 1μmol L‐1) in organ chambers for measurement of isometric tension.ResultsThe endothelium‐dependent relaxation induced by ACh was decreased in CSM from ethanol, diabetic, ethanol‐diabetic groups when compared with the control group. Immunohistochemistry for eNOS, iNOS, ETA eeeand ETB showed an increase in cavernosal smooth muscle cells in the ethanol, diabetic and ethanol‐diabetic groups when compared with the control group. Similarly, the mRNA levels for eNOs, ETA eeeand ETB were increased in CSM of groups ethanol, diabetic and ethanol‐diabetic and miRNAs‐27a, 155 and 199 were decreased in cavernosal smooth muscle from the ethanol, diabetic and ethanol‐diabetic groups.ConclusionsOur results showed that chronic ethanol consumption associated with diabetes was an impairment of relaxation of CSM from ethanol, diabetic, and ethanol‐diabetic rats that involved a decrease in the NO pathway by endothelium‐dependent mechanisms accompanied by a change in the corpus cavernosum contractile sensitivity. We can also suggest that chronic ethanol consumption associated with diabetes changed the endothelinergic system in the CSM played a role in the pathogenesis of erectile dysfunction, suggesting that these changes of endothelin receptors were regulated by miRNAs‐155, 199.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Temporal requirement of Nkx2-5 during cardiac conduction system development

During cardiac development, transient invaginations of the myocardium, termed trabeculae, appear at the inner surface of the ventricles, which disappeared by compaction to form a functional ventricular wall. Trabeculae also contain progenitor cells of the ventricular conduction system (VCS), a complex network of Purkinje fibers driving the rapid propagation of electrical activity in the ventricles. Defects in ventricular compaction and conduction have been observed in patients and mutant mice carrying mutations in NKX2-5, encoding a key transcriptional regulator of heart development. In order to analyze the link between trabecular fate and VCS differentiation, we carried out genetic tracing and prospective clonal analyses of trabeculae using Cx40-CreERT2 mice and Rosa26-Confetti mice. Our results show that the peripheral VCS segregates progressively during embryonic development. Cells exclusively fated to give rise to the VCS are present in the trabecular compartment as early as E9.5. Specification of new conductive myocytes within the trabecular compartment during subsequent development contributes to the formation of a complex Purkinje fiber network at birth. We performed the same lineage tracing experiments in Nkx2-5 heterozygous mice with severe hypoplasia of the Purkinje fiber network at adult stages. The number of Purkinje fibers originating from early Nkx2-5 hapolinsufficient progenitors is identical to control. In contrast, at later stages of development, we observed an increase in the number of trabecular cells that do not give rise to Purkinje fibers. This suggests that Nkx2-5 controls the progressive recruitment of trabecular cells into the VCS as well as the later maintenance of the conductive phenotype. Overall our study highlights the early segregation of the VCS lineage at the onset of trabeculation and the later role of Nkx2-5 in formation of the Purkinje fiber network.

Expression profile of endothelin receptors (ETA and ETB) and microRNAs-155 and -199 in the corpus cavernosum of rats submitted to chronic alcoholism and diabetes mellitus.

Recent evidence shows that chronic ethanol consumption increases endothelin (ET)-1 induced sustained contraction of trabecular smooth muscle cells of the corpora cavernosa in corpus cavernosum of rats by a mechanism that involves increased expression of ETA and ETB receptors. Our goal was to evaluate the effects of alcohol and diabetes and their relationship to miRNA-155, miRNA-199 and endothelin receptors in the corpus cavernosum and blood of rats submitted to the experimental model of diabetes mellitus and chronic alcoholism. Forty-eight male Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D), and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study the protein expression of endothelin receptors by immunohistochemistry and expression of miRNAs-155 and -199 in serum and the cavernous tissue. Immunostaining for endothelin receptors was markedly higher in the A, D, and AD groups than in the C group. Moreover, a significant hypoexpression of the miRNA-199 in the corpus cavernosum tissue from the AD group was observed, compared to the C group. When analyzing the microRNA profile in blood, a significant hypoexpression of miRNA-155 in the AD group was observed compared to the C group. The miRNA-199 analysis demonstrated significant hypoexpression in D and AD groups compared to the C group. Our findings in corpus cavernosum showed downregulated miRNA-155 and miRNA-199 levels associated with upregulated protein expression and unaltered mRNA expression of ET receptors suggesting decreased ET receptor turnover, which can contribute to erectile dysfunction in diabetic rats exposed to high alcohol levels.

Effects of atorvastatin on porcine aqueous humour outflow and trabecular meshwork cells.

Primary open-angle glaucoma (POAG) with complex pathogenesis is one of the many major causes of blindness. It is widely accepted that the major cause of POAG is the dysregulation of the trabecular meshwork (TM), which regulates the resistance to aqueous humour outflow. Intraocular pressure is elevated with increasing outflow resistance in the conventional pathway, which consists of the TM and Schlemm's canal. The TM is a filter made up of extracellular matrix (e.g., collagens), most of which is organized into a network of beams covered by endothelial-like trabecular cells. Currently, lack of effective anti-glaucoma drugs acting on TM to normalize trabecular outflow represents a bottleneck for POAG therapy. Atorvastatin, a lipid-lowering drug, has been proven to be of benefit for POAG. The present study aimed to investigate the possible mechanisms of action of atorvastatin on the TM by using a porcine aqueous humour outflow model in vivo and TM cells in vitro. Perfusion of enucleated porcine eyes with atorvastatin (50–200 µM) for 2 h increased aqueous humour outflow (P<0.05, n=6), possibly via regulating the morphology of TM cells and the distribution of the cytoskeleton. Atorvastatin decreased adhesion molecules at the mRNA and protein level. No cytotoxicity of atorvastatin on TM cells was observed at concentrations of <100 µM. The atorvastatin-induced effects mentioned above were reversible after removal of the compound only if the atorvastatin concentration was <100 µM. The present study demonstrated that atorvastatin efficaciously elevated aqueous humour outflow, possibly due to affecting TM-cell morphology, cytoskeleton and cell junctions. Statins may be potential therapeutic agents for lowering intraocular pressure in POAG.

Special Issue on “Additive Manufacturing Technologies and Applications”

Additive Manufacturing (AM) is a well-known technology, first patented in 1984 by the French scientist Alain Le Mehaute [...]

Enzymatic Activity of CYP1B1 in primary congenital glaucoma goniodysgenesis and its relation with histological alterations

PurposeTo analyze de CYP1B1 genotype activity, the histological findings and the clinical phenotypes in human primary congenital glaucoma (PCG).MethodsTransmission electron microscopy was used to study surgical pieces from trabeculectomy from PCG patients (n = 5) and normal cadaver donors sclerocorneal rims (n = 3). Genomic DNA was extracted from the peripheral leukocytes of the same patients for the CYP1B1 study.ResultsThe histological analysis allowed us to classified patient samples into three groups depending on goniodysgenesis findings: A) no visualization of the Schlemm's canal (SC); B) presence of the SC and no visualization of the collector channels (CC); C) presence of the SC and CC. Patients from groups A and B had extensive trabecular endothelial cell necrosis, and abnormal trabecular beam and null enzymatic activity of CYP1B1. Patients from group C in addition to presence of the SC and CC, showed a more developed trabecular meshwork than groups A and B and trabecular endothelial cells alive and with signs of phagocytic. This group showed a 60% of enzymatic activity of CYP1B1. All groups shared an abnormal insertion of the ciliary muscle although in group C the insertion was in a more posterior position.ConclusionsIn the samples analyzed, it seems to have a relationship between enzymatic activity of CYP1B1 and histological changes. The lower the CYP1B1 gene enzymatic activity, the greater the goniodisgenesys and the trabecular endothelial cell death extent. A greater understanding of histological changes associated with the enzymatic activity of CYP1B1 could be useful for clinicians to plan treatment strategy. This data could be useful for clinicians to plan treatment strategy

Merkel Cell Carcinoma of Cheek: 2 Case Reports

Merkel cell carcinoma (MCC) is a rare aggressive skin cancer with the mortality rate of 33%. MCC was first described by Toker in 1972 as a trabecular cell carcinoma and is classified as a neuroendocrine tumor similar to small cell lung carcinoma and melanoma. The pathogenesis of MCC remains largely unknown. However, ultraviolet radiation and immunosuppression are likely to play a significant pathogenic role. The primary skin lesion is usually asymptomatic and it typically presents as a red or purple dome-shaped nodule. The management of MCC is controversial, however, local wide excision followed by radiotherapy is accepted as the primary treatment modality. The regional draining nodal basin is the most common site for recurrence. Therefore, sentinel lymph node biopsy is recommended in all cases, except for the clinically node-negative cases. We herein report two cases of MCC on the left cheek with different clinical manifestations. Key words: Cheek ㆍ Merkel cell carcinoma ㆍ Neoplasms

A Robust Multifunctional Sandwich Panel Design with Trabecular Structures by the Use of Additive Manufacturing Technology for a New De-Icing System

Anti-ice systems assure a vital on-board function in most aircraft: ice prevention or de-icing is mandatory for all aerodynamic surfaces to preserve their performance, and for all the movable surfaces to allow the proper control of the plane. In this work, a novel multi-functional panel concept which integrates anti-icing directly inside the primary structure is presented. In fact, constructing the core of the sandwich with trabecular non-stochastic cells allows the presence of a heat exchanger directly inside the structure with a savings in weight and an improvement in thermal efficiency. This solution can be realized easily in a single-piece component using Additive Manufacturing (AM) technology without the need for joints, gluing, or welding. The objective of this study is to preliminarily investigate the mechanical properties of the core constructed with Selective Laser Melting (SLM); through the Design of Experiment (DOE), different design parameters were varied to understand how they affect the compression behaviour.

Observation of impact energy absorption performance on idealised trabecular forms in laser sintered nylon

Purpose This paper aims to investigate whether the trabecular architecture found in natural bone can be effectively replicated through the selective laser sintering process of Nylon P2200. Design/methodology/approach Trabecular bone was idealised into a scaled up hexagonal cell proven to replicate the natural structure. The structure was modelled in Solidworks 2013 to form a network of interlinking cells. The specific property analysed was the structure toughness through the measurement of the energy absorbed before sample fracture. Findings It was found that the impact absorption can be increased with the integration of a greater number of trabecular cells producing a finer resolution and not necessarily by increasing the trabecular size. The information gained from this research may be useful in the design of impact and shock absorbing components, with an emphasis on efficient use of material mass. Research limitations/implications Designers and engineers may find biomimetic methods of absorbing shock and impact an efficient alternative consideration in design applications. Practical implications The trabecular architecture should be designed so as to be weaker than the bounding surfaces, ensuring that the individual trabecular experience failure first, maximising their energy absorbing capability through increasing the period of deceleration. The simplest way of doing this is to ensure the rod thickness is less than the bounding material thickness. Originality/value This work documents original testing of both the RP material and consolidated design of samples of idealised bone structures. It builds on previous work in the area and through the results of empirical testing, derives recommendations for further considerations in this area of design and manufacture of biomimetic structures.

3D map of the human corneal endothelial cell.

Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions.

Canonical FGFs Prevent Osteogenic Lineage Commitment and Differentiation of Human Bone Marrow Stromal Cells Via ERK1/2 Signaling.

Controlling the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) in favor of osteogenesis represents a promising approach for osteoporosis therapy and prevention. Previously, Fibroblast Growth Factor 1 (FGF1) and its subfamily member FGF2 were scored as leading candidates to exercise control over skeletal precursor commitment and lineage decision albeit literature results are highly inconsistent. We show here that FGF1 and 2 strongly prevent the osteogenic commitment and differentiation of hBMSCs. Mineralization of extracellular matrix (ECM) and mRNA expression of osteogenic marker genes Alkaline Phosphatase (ALP), Collagen 1A1 (COL1A1), and Integrin-Binding Sialoprotein (IBSP) were significantly reduced. Furthermore, master regulators of osteogenic commitment like Runt-Related Transcription Factor 2 (RUNX2) and Bone Morphogenetic Protein 4 (BMP4) were downregulated. When administered under adipogenic culture conditions, canonical FGFs did not support osteogenic marker expression. Moreover despite the presence of osteogenic differentiation factors, FGFs even disabled the pro-osteogenic lineage decision of pre-differentiated adipocytic cells. In contrast to FGF Receptor 2 (FGFR2), FGFR1 was stably expressed throughout osteogenic and adipogenic differentiation and FGF addition. Moreover, FGFR1 and Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) were found to be responsible for underlying signal transduction using respective inhibitors. Taken together, we present new findings indicating that canonical FGFR-ERK1/2 signaling entrapped hBMSCs in a pre-committed state and arrested further maturation of committed precursors. Our results might aid in unraveling and controlling check points relevant for ageing-associated aberrant adipogenesis with consequences for the treatment of degenerative diseases such as osteoporosis and for skeletal tissue engineering strategies. J. Cell. Biochem. 118: 263-275, 2017. © 2016 Wiley Periodicals, Inc.

Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition

Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.

Combined bone internal and external remodeling based on Eshelby stress

Models for the internal and external bone remodeling are developed in the framework of the thermodynamics of irreversible processes. Internal remodeling is essentially accounted for by an evolution of the internal bone density versus the trace of the Eshelby stress. The growing surface is endowed with a specific mechanical behavior elaborated from a surface potential, depending upon the elastic surface strain and the external normal vector. The surface remodeling velocity is related to the driving force for growth identified as the surface divergence of Eshelby stress. The developed formalism is applied to simulate bone internal and external remodeling for 2D geometries, showing the influence of external mechanical stimuli on the evolution of the external shape of bone. Simulations of the evolution of the shape and density of bone structures are carried out successively at mesoscopic, microscopic and macroscopic levels, the former representing cellular level simulations of the trabecular unit cell. At the macroscopic level, a linear elastic constitutive law for trabecular bone is derived, relying on the discrete homogenization approach, whereby the continuum parameters such as stiffness evolve with morphology as remodeling occurs within bone. The developed numerical platform is able to simulate combined bone internal and external remodeling at both trabecular and macroscopic scales.

Tadao Nakago, 1933-2016

Tadao Nakago died of bladder cancer on February 12, 2016, just 2 days before his 83rd birthday. He was born on February 14, 1933, in Sanda, north of the Rokko Mountains in Kobe, Japan. He graduated from Osaka University Dental School in 1957 and became a dentist. He chose orthodontics as his specialty and earned a PhD degree from Osaka University in 1965. His academic interest in the early phase of his career was the cephalometric assessment of craniofacial development in the Japanese population to qualify desirable tooth movement. He became a founding professor of orthodontics at Matsumoto Dental College in 1972. He moved to Okayama University in 1980 as a founding director of the dental school-affiliated hospital and served as president of the hospital until 1988. In 1982, he successfully moved the government of Japan to include, for the first time, orthodontic treatment for craniofacial anomalies, such as cleft palate, on the list for national health insurance reimbursement. He served as dean of the dental school from 1992 to 1994. He survived a bitter administrative storm at Okayama University in 1996 and restored its honor at the law court in 2002. He loved to educate young people and tried to disseminate practical thinking about the standards of orthodontic treatment and care. He translated Graber's Textbook of Orthodontics into Japanese, and it became a best-seller. He liked to organize laboratory studies to explain the mechanism of tooth movement. He designed a device to apply cyclic mechanical stretching to cultured cells in a Petri dish.1Matsuo T. Uchida H. Matsuo T. Bovine and porcine trabecular cells produce prostaglandin F2α in response to cyclic mechanical stretching.Jpn J Ophthalmol. 1996; 40: 289-296PubMed Google Scholar The cell-stretching machine was manufactured in 1983 at the workshop affiliated with the Faculty of Engineering of Okayama University. This unique original device preceded commercially available apparatuses and contributed to new knowledge in other fields of medicine.2Okada Y. Matsuo T. Ohtsuki H. Bovine trabecular cells produce TIMP-1 and MMP-2 in response to mechanical stretching.Jpn J Ophthalmol. 1998; 42: 90-94Crossref Scopus (44) Google Scholar, 3Sato Y. Matsuo T. Ohtsuki H. A novel gene (oculomedin) induced by mechanical stretching in human trabecular cells of the eye.Biochem Biophys Res Commun. 1999; 259: 349-351Crossref Scopus (30) Google Scholar, 4Yamaguchi K. Matsuo T. Shiraga F. Ohtsuki H. TIMP-1 production by bovine retinal pigment epithelial cells increases in response to cyclic mechanical stretch.Jpn J Ophthalmol. 2001; 45: 470-474Crossref Scopus (5) Google Scholar, 5Yamaoka A. Matsuo T. Shiraga F. Ohtsuki H. TIMP-1 production by human scleral fibroblast decreases in response to cyclic mechanical stretching.Ophthalmic Res. 2001; 33: 98-101Crossref Scopus (22) Google Scholar, 6Namba M. Matsuo T. Shiraga F. Ohtsuki H. Retinal cells produce TIMP-1 and TIMP-2 in response to cyclic mechanical stretching.Ophthalmic Res. 2001; 33: 163-169Crossref Scopus (11) Google Scholar His last work was focused on the threshold of hydraulic pressure to induce cellular response in periodontal ligament fibroblasts.7Nakago-Matsuo C. Matsuo T. Nakago T. Intracellular calcium response to hydraulic pressure in human periodontal ligament fibroblasts.Am J Orthod Dentofacial Orthop. 1996; 109: 244-248Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 8Nakago-Matsuo C. Matsuo T. Nakago T. Basal nitric oxide production is enhanced by hydraulic pressure in cultured human periodontal ligament fibroblasts.Am J Orthod Dentofacial Orthop. 2000; 117: 474-478Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar The threshold value was consistent with the force needed to move teeth in a clinical orthodontic setting. The work was continued by his daughter, an orthodontist, in collaboration with her husband, an ophthalmologist.9Matsuo T. Matsuo N. Intracellular calcium response to hydraulic pressure in human trabecular cells.Br J Ophthalmol. 1996; 80: 561-566Crossref Scopus (27) Google Scholar, 10Matsuo T. Basal nitric oxide production is enhanced by hydraulic pressure in cultured human trabecular cells.Br J Ophthalmol. 2000; 84: 631-635Crossref Scopus (15) Google Scholar Dr Nakago's funeral was held on his 83rd birthday, with family and friends present at the Buddhist temple where his wife's father had served as a priest.

Differentially Expressed MicroRNAs in Meningiomas Grades I and II Suggest Shared Biomarkers with Malignant Tumors.

Meningiomas represent the most common primary tumors of the central nervous system, but few microRNA (miRNA) profiling studies have been reported so far. Deep sequencing of small RNA libraries generated from two human meningioma biopsies WHO grades I (benign) and II (atypical) were compared to excess dura controls. Nineteen differentially expressed miRNAs were validated by RT-qPCR using tumor RNA from 15 patients and 5 meninges controls. Tumor suppressor miR-218 and miR-34a were upregulated relative to normal controls, however, miR-143, miR-193b, miR-451 and oncogenic miR-21 were all downregulated. From 10 selected putative mRNA targets tested by RT-qPCR only four were differentially expressed relative to normal controls. PTEN and E-cadherin (CDH1) were upregulated, but RUNX1T1 was downregulated. Proliferation biomarker p63 was upregulated with nuclear localization, but not detected in most normal arachnoid tissues. Immunoreactivity of E-cadherin was detected in the outermost layer of normal arachnoids, but was expressed throughout the tumors. Nuclear Cyclin D1 expression was positive in all studied meningiomas, while its expression in arachnoid was limited to a few trabecular cells. Meningiomas of grades I and II appear to share biomarkers with malignant tumors, but with some additional tumor suppressor biomarkers expression. Validation in more patients is of importance.