Physical limitations to tissue engineering of intervertebral disc cells: effect of extracellular osmotic change on glycosaminoglycan production and cell metabolism. Laboratory investigation

OBJECT: In this study, the authors examined how physiological levels of extracellular osmolality influence proteoglycan accumulation in nucleus pulposus cells in a 3D culture system. METHODS: Cells were isolated from the nucleus pulposus of caudal discs obtained from 18- to 24-month-old bovines. They were cultured for 6 days in alginate beads at 4 million cells/ml in Dulbecco modified Eagle medium containing 6% fetal bovine serum under 21% O2. Medium osmolality was altered by NaCl addition between 270 and 570 mOsm and monitored using a freezing point osmometer. The cell viability profile was determined by manual counting after trypan blue staining. Profiles across intact beads were determined by manual counting by using fluorescent probes and a transmission electron microscope. Lactate production was measured enzymatically, and glycosaminoglycan (GAG) accumulation was measured using a dimethylmethylene blue assay. Rate of sulfate GAG synthesis was measured using a standard [35S]sulfate radioactive method. RESULTS: The cell viability was similar for the high- and low-osmolality cultures. However, confocal microscopy showed that the cells were the largest at 270 mOsm and became smaller with increasing osmotic pressure. The GAG production was largest at 370 mOsm, the capacity for GAG production and cell metabolism (lactate production) was low under hypoosmolality and hyperosmolality, and cell death was observed on electron microscopy. CONCLUSIONS: In the authors' model, the prevailing osmolality was a powerful regulator of GAG accumulation by cultured nucleus cells. Thus, these results indicate that GAG synthesis rates are regulated by GAG concentration, with implications both for the cause of degeneration and for tissue engineering.     

Structural insight into the membrane insertion of tail‐anchored proteins by Get3

Tail anchored (TA) proteins, which are important for numerous cellular processes, are defined by a single transmembrane domain (TMD) near the C‐terminus. The membrane insertion of TA proteins is mediated by the highly conserved ATPase Get3. Here we report the crystal structures of Get3 in ADP‐bound and nucleotide‐free forms at 3.0 Å and 2.8 Å resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn²⁺‐mediated homodimer in a head‐to‐head orientation, representing an open dimer conformation. Our cross‐link experiments indicated the closed dimer‐stimulating ATP hydrolysis, which might be coupled with TA‐protein release. Further, our coexpression‐based binding assays using a model TA protein Sec22p revealed the direct interaction between the helical domain of Get3 and the Sec22p TMD. This interaction is independent of ATP and dimer formation. Finally, we propose a structural mechanism that links ATP hydrolysis with the TA‐protein insertion mediated by the conserved DTAPTGH motif.     

Continuing Medical Education Activity in Echocardiography

Article Title: Speckle Tracking Global Strain Rate E/E’ Predicts LV Filling Pressure More Accurately Than Traditional Tissue Doppler E/E’ (Echocardiography, 2012;29:403)     

Bactericidal activity of cefclidin (E1040) against Pseudomonas aeruginosa under conditions simulating plasma pharmacokinetics: lack of development of chromosomally-mediated resistance to beta-lactams.

The bactericidal activity of cefclidin (E1040), a new cephalosporin, against Pseudomonas aeruginosa was compared with that of ceftazidime and imipenem in an in-vitro model in which antibiotic concentration was varied continuously. A two-compartment open in-vitro model was used to simulate the plasma pharmacokinetics of each antibiotic in man for 12 h after a 1 h infusion of 1 g iv. The bactericidal activity of each antibiotic was observed for 6 h; however, it was diminished or absent after 6 h when the antibiotic concentration fell near to the MIC. With ceftazidime and imipenem, marked regrowth was observed after 6 h. Moreover, selection of resistant variants was observed with ceftazidime, and these variants produced 200 to 500 times more beta-lactamase than the corresponding wild-type strains. With cefclidin neither marked regrowth nor emergence of resistant variants was observed. The affinity of cefclidin for the chromosomal beta-lactamase produced by P. aeruginosa was much lower than the affinities of other new beta-lactams, and cefclidin was hydrolyzed more slowly than ceftazidime at a low concentration (2 microM). The high activity of cefclidin against P. aeruginosa, which results mainly from the low affinity of cefclidin for the pseudomonal beta-lactamase, and may play a major role in the absence of regrowth and lack of selection of resistant variants.     

Effect of upper gastrointestinal endoscopy on circulation in the elderly.

BACKGROUND: Upper gastrointestinal endoscopy in the elderly is increasingly becoming more common, despite the possibility that a minimal load on the circulation can cause serious complications such as shock and cardiac arrest. OBJECTIVE: The effects of endoscopy on the heart and the possibility of predicting circulatory accidents were studied using natriuretic peptide levels. METHODS: The patients were randomly chosen according to their age and divided into an elderly group (over 60 years of age, 64 patients) and a young group (under 30 years of age, 20 patients). The patients in the elderly group were further subdivided into two groups based on the presence or absence of circulatory complications (46 patients with circulatory complications and 18 without complications). The load on the heart was evaluated by measuring human atrial natriuretic peptide (hANP) and human brain natriuretic peptide (hBNP) which are secreted by the myocardial cells in response to cardiac load. Specimens were obtained before and after endoscopy. RESULTS: The hANP level was significantly higher after endoscopy in the elderly group, regardless of the presence or absence of circulatory complications. No significant difference was observed in the hBNP level. No significant increase in hANP or hBNP levels was observed after endoscopy in the young group. CONCLUSIONS: These observations suggest an increased atrial load during endoscopy in the elderly. The increase in pulse rate during endoscopy is one possible cause of atrial load. Therefore, the risk of circulatory system damage must be recognized when endoscopy is performed in the elderly. The measurement of plasma hANP and hBNP levels may provide effective indices for evaluating cardiac load during endoscopy.     

A CASE OF CHOLESTEROSIS OF THE COMMON BILE DUCT DIAGNOSED BY PERORAL CHOLANGIOSCOPY

We experienced a case of cholesterosis of the common bile duct in which peroral cholangioscopy (POCS) was very useful for making a diagnosis. The patient was a 66‐year‐old man. He was admitted for examination of an asymptomatic mass in the pancreas. The mass was diagnosed as a serous cystic tumor of the pancreas. While examining the tumor, endoscopic retrograde cholangiopancreatography (ERCP) incidentally revealed a 7 mm irregular filling defect in the distal common bile duct. EST was then performed, followed by POCS, which revealed a clump of yellowish‐white Ip or Isp polyps in the distal bile duct. Histological examination of biopsy specimens taken under direct vision gave a diagnosis of cholesterol polyps containing foamy cells. POCS was useful for allowing direct observation of the characteristic color of cholesterol polyps, and for obtaining biopsy specimens under direct vision.     

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.