Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.      

DNA repair efficiency for ultraviolet induced damage

Ultraviolet radiation causes lesions in bacterial DNA which are repaired by several enzyme systems. Wide variations in the efficiency of repair for differentE. Coli strains are inadequately explained by a simple presence or absence of one or more repair systems. It is proposed that a major factor in the variations is the sensitivity of the repair systems themselves to ultraviolet induced interactions between proteins and the repair enzyme cistrons. An analytic approach is applied to pre-existing data to establish the numbers of thymine and cytosine bases in the repair cistrons, lending support to the model. The findings imply that bacteria will become sensitive to UV upon inhibition of one of four amino acids.     

Cardiac assist with a twist: apical torsion as a means to improve failing heart function

Changes in muscle fiber orientation across the wall of the left ventricle (LV) cause the apex of the heart to turn 10-15 deg in opposition to its base during systole and are believed to increase stroke volume and lower wall stress in healthy hearts. Studies show that cardiac torsion is sensitive to various disease states, which suggests that it may be an important aspect of cardiac function. Modern imaging techniques have sparked renewed interest in cardiac torsion dynamics, but no work has been done to determine whether mechanically augmented apical torsion can be used to restore function to failing hearts. In this report, we discuss the potential advantages of this approach and present evidence that turning the cardiac apex by mechanical means can displace a clinically significant volume of blood from failing hearts. Computational models of normal and reduced-function LVs were created to predict the effects of applied apical torsion on ventricular stroke work and wall stress. These same conditions were reproduced in anesthetized pigs with drug-induced heart failure using a custom apical torsion device programmed to rotate over various angles during cardiac systole. Simulations of applied 90 deg torsion in a prolate spheroidal computational model of a reduced-function pig heart produced significant increases in stroke work (25%) and stroke volume with reduced fiber stress in the epicardial region. These calculations were in substantial agreement with corresponding in vivo measurements. Specifically, the computer model predicted torsion-induced stroke volume increases from 13.1 to 14.4 mL (9.9%) while actual stroke volume in a pig heart of similar size and degree of dysfunction increased from 11.1 to 13.0 mL (17.1%). Likewise, peak LV pressures in the computer model rose from 85 to 95 mm Hg (11.7%) with torsion while maximum ventricular pressures in vivo increased in similar proportion, from 55 to 61 mm Hg (10.9%). These data suggest that: (a) the computer model of apical torsion developed for this work is a fair and accurate predictor of experimental outcomes, and (b) supra-physiologic apical torsion may be a viable means to boost cardiac output while avoiding blood contact that occurs with other assist methods.     

Mechanistic analysis of a DNA damage-induced, PTEN-dependent size checkpoint in human cells

Following DNA damage, human cells undergo arrests in the G(1) and G(2) phases of the cell cycle and a simultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupled from the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we show that the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizing radiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutational analysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylation is unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectors involved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach, which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex that includes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN(+/+) cells recapitulated the lack of size checkpoint control seen in PTEN(-/-) cells. Taken together, these results provide further support for the existence of a DNA damage-inducible size checkpoint that is regulated by a major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controls cell size.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Early Tertiary fossil plants and paleoclimate of Lanzhou Basin

Fossil plants from the lower part of Xianshuihe Formation in the Lanzhou Basin, Gansu Province were studied. The flora contains 29 species, representing 20 genera and 12 families, which include Lauraceae ( Daphnogene ), Lardizabalaceae ( Akebia ), Berberidaceae ( Berberis ), Ulmaceae ( Planera, Ulmus, Zelkova ), Betulaceae ( Alnus, Carpinus ), Myricaceae( Myrica ), Salicaceae ( Populus, Salix), Myrsinaceae(Ardisia), Rosaceae ( Prunus, Sorbus, Sorbaria, Spiraea ), Leguminosae ( Gleditsia, Sophora), Anacardiaceae (Rhus), Caprifoliaceae(Viburnum). An analysis of the floristic elements and their foliar physiognomy shows that most members of the flora are deciduous broad-leaved trees or shrubs with a few evergreen shrubs. The most noteworthy species is Rhus turcomanica which was present in the Middle Eocene to Late Eocene of Central Asia (Kazakhstan, Turkmenistan). Generally, Rhus turcomanica occurred at the same beds as Palibinia, an extinct fossil plant whose presence indicates a subtropical dry climate. Another species, Sorbaria callicomifolia Kornilova was present from the Early Oligocene to Early Miocene of Central Asia (Kazakhstan and Turkmenistan). According to an analysis of spores and pollen, this flora contains over 20 species. It is predominated by the angiosperm pollen. There appeared Ephedripites and Nitrariadites which were important elements in the dry area. Ephedripites was found from the Upper Cretaceous to Early Tertiary. Nitrariadites occurred in the Late Miocene, whereas Rhus turcomanica and Sorbaria callicomifolia were both reported in the subtropical dry area from the Middle Eocene to Early Oligocene. The latest record of Rhus turcomanica is from the Middle Eocene to Early Oligocene of Central Asia. The presence of this element in the lower part of Xianshuihe Formation may indicate that itsage is the latest stage of the Early Oligocene.     

Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations

We have developed a fluorescence-based single strand conformation polymorphism (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer for unique color detection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell lines with reported mutations in p53 exons 5-8 were analyzed by SSCP using various gel conditions. The sensitivity for mutation detection was 95% for all cell lines studied, and no false positives were seen in 10 normal DNA samples for all four exons. Experiments mixing known amounts of tumor and normal DNA showed that mutations were detected even when tumor DNA was mixed with 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a useful tool in cancer research, where screening large numbers of samples for p53 mutations is desired.