Biomaterials for the programming of cell growth in oral tissues: The possible role of APA

Examples of programmed tissue response after the interaction of cells with biomaterials are a hot topic in current dental research. We propose here the use of anodic porous alumina (APA) for the programming of cell growth in oral tissues. In particular, APA may trigger cell growth by the controlled release of specific growth factors and/or ions. Moreover, APA may be used as a scaffold to promote generation of new tissue, due to the high interconnectivity of pores and the high surface roughness displayed by this material.     

Production of an auto-stimulatory growth factor by human hepatoma cells abrogates requirement for a brain-derived factor

Summary Human hepatoma cells grow at high cell density in the absence of exogenous growth factors. At low cell density, two different hepatoma cell lines required a novel growth factor from brain tissue. A factor with similar physico-chemical properties in the concentrated medium from high density cultures completely substituted for the brain extract. The autogenous secretion of a novel liver cell growth factor that is concentrated in brain tissue may underlie in part the unregulated growth of hepatomas. EDITOR'S STATEMENT Collective properties of growth factor activities for hepatoma cells in bovine brain extract and the medium of hepatoma cells suggest a neural tissue-derived liver cell growth factor that may be autogenously produced by liver tumor cells. Purification and characterization of such a factor may lead to selective inhibition of hepatoma, cell proliferation. David W. Barnes     

Insulin secretion dynamics of free and alginate-encapsulated insulinoma cells

This study investigates the effect of alginate/poly-l-lysine/alginate (APA) encapsulation on the insulin secretion dynamics exhibited by an encapsulated cell system. Experiments were performed with the aid of a home-built perfusion apparatus providing a 1 min temporal resolution. Insulin profiles were measured from: (i) murine insulinoma βTC3 cells encapsulated in calcium alginate/poly-l-lysine/alginate (APA) beads generated with high guluronic (G) or high mannuoric (M) content alginate, and (ii) murine insulinoma βTC-tet cells encapsulated in high M APA beads and propagated in the presence and absence of tetracycline. Results show that encapsulation in APA beads did not affect the insulin secretion profile shortly post-encapsulation. However, remodeling of the beads due to cell proliferation affected the insulin secretion profiles; and inhibiting remodeling by suppressing cell growth preserved the secretion profile. The implications of these findings regarding the in vivo function of encapsulated insulin secreting cells are discussed.     

Mitogenic factors of corpora lutea

The mammalian corpus luteum (CL), which plays a central role in the reproductive process because of its production of hormones such as progesterone, appears to be an exceptionally dynamic organ. Its rate of growth and development are extremely rapid and, even when the CL is functionally mature, its rate of cell turnover remains relatively high. Associated with this high rate of cell turnover, the mature CL receives the greatest blood supply per unit tissue of any organ, and also exhibits a relatively high metabolic rate. Although numerous growth factors have been identified in luteal tissue, their role in growth and differentiation of this dynamic organ remains unclear. Recently, while attempting to identify mitogenic factors of ovine and bovine CL, we have found that they produce several mitogens during the estrous cycle as well as pregnancy. The majority of these luteal-derived mitogenic factors are heparin-binding, and although some may represent previously identified factors, several appear to be novel heparin-binding growth factors. Isolation and purification of mitogenic factors produced by the CL will enable us to determine their roles in luteal growth, development and differentiated function, which will contribute to our understanding not only of the regulation of fertility but also of tissue growth and development in general.     

Effect of cell seeding and mechanical loading on vascularization and tissue formation inside a scaffold: A mechano-biological model using a lattice approach to simulate cell activity

Achieving successful vascularization remains one of the main problems in bone tissue engineering. After scaffold implantation, the growth of capillaries into the porous construct may be too slow to provide adequate nutrients to the cells in the scaffold interior and this inhibits tissue formation in the scaffold core. Often, prior to implantation, a controlled cell culture environment is used to stimulate cell proliferation and, once in place, the mechanical environment acting on the tissue construct is determined by the loading conditions at the implantation site. To what extent do cell seeding conditions and the construct loading environment have an effect on scaffold vascularization and tissue growth? In this study, a mechano-biological model for tissue differentiation and blood vessel growth was used to determine the influence of cell seeding on vascular network development and tissue growth inside a regular-structured bone scaffold under different loading conditions. It is predicted that increasing the number of cells seeded homogeneously reduces the rate of vascularization and the maximum penetration of the vascular network, which in turn reduces bone tissue formation. The seeding of cells in the periphery of the scaffold was predicted to be beneficial for vascularization and therefore for bone growth; however, tissue formation occurred more slowly during the first weeks after implantation compared to homogeneous seeding. Low levels of mechanical loading stimulated bone formation while high levels of loading inhibited bone formation and capillary growth. This study demonstrates the feasibility of computational design approaches for bone tissue engineering.     

Hybrid cellular automaton modeling of nutrient modulated cell growth in tissue engineering constructs

Mathematic models help interpret experimental results and accelerate tissue engineering developments. We develop in this paper a hybrid cellular automata model that combines the differential nutrient transport equation to investigate the nutrient limited cell construct development for cartilage tissue engineering. Individual cell behaviors of migration, contact inhibition and cell collision, coupled with the cell proliferation regulated by oxygen concentration were carefully studied. Simplified two-dimensional simulations were performed. Using this model, we investigated the influence of cell migration speed on the overall cell growth within in vitro cell scaffolds. It was found that intense cell motility can enhance initial cell growth rates. However, since cell growth is also significantly modulated by the nutrient contents, intense cell motility with conventional uniform cell seeding method may lead to declined cell growth in the final time because concentrated cell population has been growing around the scaffold periphery to block the nutrient transport from outside culture media. Therefore, homogeneous cell seeding may not be a good way of gaining large and uniform cell densities for the final results. We then compared cell growth in scaffolds with various seeding modes, and proposed a seeding mode with cells initially residing in the middle area of the scaffold that may efficiently reduce the nutrient blockage and result in a better cell amount and uniform cell distribution for tissue engineering construct developments.     

Homogeneous cultivation of animal cells for the production of virus and virus products

Homogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used. A microcarrier method in which cells adhere to DEAE-Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem. Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 10⁷ plaque forming units per milliliter for over 50 days with Rubella-infected BHK cells.     

Aminopeptidase-A. I. CDNA cloning and expression and localization in rat tissues

Aminopeptidase-A (APA) is an ectoenzyme that selectively hydrolyzes acidic residues from the amino terminus of oligopeptides, including biologically active [Asp(1)]ANG II and [Asp(1)]CCK-8. We sought to characterize rat APA by cDNA cloning and expression and to determine its tissue distribution by in situ hybridization and immunohistochemistry. Sequence analysis of overlapping cDNA clones isolated from rat kidney cDNA libraries indicated that the full-length cDNA encoded a 945-amino acid protein with a predicted molecular mass of 108 kDa; the size was confirmed by in vitro translation of a full-length cDNA construct. Transient transfection of the full-length cDNA construct in mammalian cells yielded a protein approximately 140 kDa in size, a size that agrees with the immunoblots of APA from rat tissue and is consistent with APA being known as a glycosylated protein. Tissue APA activity and mRNA expression were highest in the kidney and ileum. Localization of APA by in situ hybridization and immunohistochemistry indicated that, with the exception of the kidney and ileum, where APA was localized to the luminal brush border of proximal tubules and enterocytes, respectively, APA was associated with either capillaries or the lining of sinusoids. Areas known to be physiological targets for ANG II, including glomeruli, the zona glomerulosa, and anterior pituitary, had high levels of APA. The localization pattern suggests that APA may subserve multiple functions, i.e., a generalized role in peptide scavenging and perhaps a more specific role in metabolism of circulating or locally produced ANG II or CCK-8.     

FACTORS AFFECTING SURFACE ALKALINE PHOSPHATASE ACTIVITY IN THE BROWN ALGA FUCUS SPIRALIS AT A NORTH SEA INTERTIDAL SITE (TYNE SANDS,SCOTLAND)1

Surface alkaline phosphatase activity (APA) was measured in the tips of Fucus spiralis L. thalli over an approximately 1-year period, using small plants from upper littoral rock pools at a site on the east coast of Scotland. Maximum APA (4.4 μmol p-NP·g dry wt^?1·h^?1) occurred in April, coincident with a sharp increase in tissue N:P ratio of the tips and the probable period of most rapid growth. Enzymatic activity was not correlated with any of the physical and chemical factors (temperature, pH, external phosphate, salinity) measured in the pools or nutrient concentrations (C, N, P) in the apical part of the thallus. No obvious correlation was found between APA and the position of a plant on the shore. However, APA was significantly higher in plants emersed at low tide than in plants submerged during the whole tidal cycle. There was a significant positive correlation between APA and tissue P within a single thallus, with the highest value at the tip (which includes the meristematic zone). Changes in APA of Fucus spiralis appear to result from a complex interaction of biotic and environmental factors, including stress by emersion, growth period, rapid changes in tissue nutrient content and N:P ratio, and regzon of thallus.     

Plasminogen is tethered with high affinity to the cell surface by the plasma protein, histidine-rich glycoprotein

Plasminogen has been implicated in extracellular matrix degradation by invading cells, but few high affinity cell surface receptors for the molecule have been identified. Previous studies have reported that the plasma protein, histidine-rich glycoprotein (HRG), interacts with plasminogen and cell surfaces, raising the possibility that HRG may immobilize plasminogen/plasmin to cell surfaces. Here we show, based on optical biosensor analyses, that immobilized HRG interacts with soluble plasminogen with high affinity and with an extremely slow dissociation rate. Furthermore, the HRG-plasminogen interaction is lysine-dissociable and involves predominately the amino-terminal domain of HRG, and the fifth kringle domain of plasminogen, but not the carboxyl-terminal lysine of HRG. HRG was also shown to tether plasminogen to cell surfaces, with this interaction being potentiated by elevated Zn(2+) levels and low pH, conditions that prevail at sites of tissue injury, tumor growth, and angiogenesis. Based on these data we propose that HRG acts as a soluble adaptor molecule that binds to cells at sites of tissue injury, tumor growth, and angiogenesis, providing a high affinity receptor for tethering plasminogen to the cell surface and thereby enhancing the migratory potential of cells.     

Fibrin gel enhanced trilayer structure in cell-cultured constructs

Efficient cell seeding and subsequent support from a substrate ensure optimal cell growth and neotissue development during tissue engineering, including heart valve tissue engineering. Fibrin gel as a cell carrier may provide high cell seeding efficiency and adhesion property, improved cellular interaction, and structural support to enhance cellular growth in trilayer polycaprolactone (PCL) substrates that mimic the structure of native heart valve leaflets. This cell carrier gel coupled with a trilayer PCL substrate may enable the production of native-like cell-cultured leaflet constructs suitable for heart valve tissue engineering. In this study, we seeded valvular interstitial cells onto trilayer PCL substrates with fibrin gel as a cell carrier and cultured them for 1 month in vitro to determine if this gel can improve cell proliferation and production of extracellular matrix within the trilayer cell-cultured constructs. We observed that the fibrin gel enhanced cellular proliferation, their vimentin expression, and collagen and glycosaminoglycan production, leading to improved structure and mechanical properties of the developing PCL cell-cultured constructs. Fibrin gel as a cell carrier significantly improved the orientations of the cells and their produced tissue materials within trilayer PCL substrates that mimic the structure of native heart valve leaflets and, thus, may be highly beneficial for developing functional tissue-engineered leaflet constructs.     

Effects of ultraviolet radiation and nutrients on the structure-function of phytoplankton in a high mountain lake

The combined effect of high solar ultraviolet radiation (UVR) and nutrient supply in a phytoplankton community of a high mountain lake is analyzed in a in situ experiment for 6 days with 2 × 2 factorial design. Interactive UVR × nutrient effects on structural and functional variables (algal biomass, chlorophyll a (chl a), primary production (PP), maximal electron transport rate (ETR(max)), and alkaline phosphatase activity (APA)), as well as stoichiometric ones (sestonic N per cell and N:P ratio) were found. Under non-nutrient enriched conditions, no deleterious effects of UVR on structural variables, PP, photosynthetic efficiency and ETR(max) were observed, whereas only particulate and total APA were affected by UVR. However, percentage excreted organic carbon (%EOC), dissolved APA and sestonic C and P per cell increased under UVR, leading to a decrease in algal C:P and N:P ratios. After nutrient enrichment, chl a, total algal biomass and PP were negatively affected by UVR whereas %EOC, ETR(max) and internal C, P and N content increased. We suggest that the mechanism of algal acclimation to UVR in this high UVR flux ecosystem seems to be related to the increase of internal algal P-content mediated by physiological mechanisms to save P and by a stimulatory UVR effect on dissolved extracellular APA. The mechanism involved in the unmasking effect of UVR after nutrient-enrichment may be the result of a greater sensitivity to UVR-induced cell damage, making the negative UVR effects more evident.     

On the genetic control of planar growth during tissue morphogenesis in plants

Tissue morphogenesis requires extensive intercellular communication. Plant organs are composites of distinct radial cell layers. A typical layer, such as the epidermis, is propagated by stereotypic anticlinal cell divisions. It is presently unclear what mechanisms coordinate cell divisions relative to the plane of a layer, resulting in planar growth and maintenance of the layer structure. Failure in the regulation of coordinated growth across a tissue may result in spatially restricted abnormal growth and the formation of a tumor-like protrusion. Therefore, one way to approach planar growth control is to look for genetic mutants that exhibit localized tumor-like outgrowths. Interestingly, plants appear to have evolved quite robust genetic mechanisms that govern these aspects of tissue morphogenesis. Here we provide a short summary of the current knowledge about the genetics of tumor formation in plants and relate it to the known control of coordinated cell behavior within a tissue layer. We further portray the integuments of Arabidopsis thaliana as an excellent model system to study the regulation of planar growth. The value of examining this process in integuments was established by the recent identification of the Arabidopsis AGC VIII kinase UNICORN as a novel growth suppressor involved in the regulation of planar growth and the inhibition of localized ectopic growth in integuments and other floral organs. An emerging insight is that misregulation of central determinants of adaxial–abaxial tissue polarity can lead to the formation of spatially restricted multicellular outgrowths in several tissues. Thus, there may exist a link between the mechanisms regulating adaxial–abaxial tissue polarity and planar growth in plants.     

Differences in polyamine metabolism between carcinomatous and uninvolved human breast tissues

The polyamine biosynthetic enzymes ODC and SAMDC show higher activity in carcinomatous human breast tissue than in uninvolved tissue of the same breast; the interconversion enzyme PAO shows significantly lower activity in carcinomatous than uninvolved tissue. The polyamine metabolism in carcinomatous human breast tissue thus appears to differ from that in uninvolved tissue. Intracellular polyamine concentrations, particularly spermine, are high in carcinomatous tissue. This increase and that of the biosynthetic enzymes suggest that a higher polyamine concentration is needed for carcinomatous cell growth. If this is the case, the lower PAO activity in carcinomatous tissue may be explained as a mechanism that conserves the high intracellular polyamine concentration.     

酸性磷酸酶法检测体外培养细胞数

利用小鼠成纤维细胞系(NIH3T3)、小鼠骨髓瘤细胞系(SP2/0)、人大肠癌细胞系(LO-VO)和人白血病细胞系(K562),评价酸性磷酸酶(APA)法用于检测体外各类型细胞的增殖和杀伤作用。用直线回归分析光吸收度与每孔活细胞数的关系。结果表明,APA法能准确地反映检测的活细胞数(相关系数均>0.99)。本方法不仅能很好地检测表皮生长因子对细胞的增殖作用,也能够检测顺铂对体外细胞的杀伤作用。结果表明APA法简单、灵敏,可以用于上皮和间质等贴壁和悬浮生长的细胞计数。     

Liver cell line derived conditioned medium enhances myofibril organization of primary rat cardiomyocytes

Cardiomyocytes are the fundamental cells of the heart and play an important role in engineering of tissue constructs for regenerative medicine and drug discovery. Therefore, the development of culture conditions that can be used to generate functional cardiomyocytes to form cardiac tissue may be of great interest. In this study, isolated neonatal rat cardiomyocytes were cultured with several culture conditions in vitro and characterized for cell proliferation, myofibril organization, and cardiac functionality by assessing cell morphology, immunocytochemical staining, and time-lapse confocal scanning microscopy. When cardiomyocytes were cultured in liver cell line derived conditioned medium without exogenous growth factors and cytokines, the cell proliferation increased, cell morphology was highly elongated, and subsequent myofibril organization was highly developed. These developed myofibril organization also showed high level of contractibility and synchronization, representing high functionality of cardiomyocytes. Interestingly, many of the known factors in hepatic conditioned medium, such as insulin-like growth factor II (IGFII), macrophage colony-stimulating factor (MCSF), leukemia inhibitory factor (LIF), did not show similar effects as the hepatic conditioned medium, suggesting the possibility of synergistic activity of the several soluble factors or the presence of unknown factors in hepatic conditioned medium. Finally, we demonstrated that our culture system could provide a potentially powerful tool for in vitro cardiac tissue organization and cardiac function study.     

Curvature- and fluid-stress-driven tissue growth in a tissue-engineering scaffold pore

Cell proliferation within a fluid-filled porous tissue-engineering scaffold depends on a sensitive choice of pore geometry and flow rates: regions of high curvature encourage cell proliferation, while a critical flow rate is required to promote growth for certain cell types. When the flow rate is too slow, the nutrient supply is limited; when it is too fast, cells may be damaged by the high fluid shear stress. As a result, determining appropriate tissue-engineering-construct geometries and operating regimes poses a significant challenge that cannot be addressed by experimentation alone. In this paper, we present a mathematical theory for the fluid flow within a pore of a tissue-engineering scaffold, which is coupled to the growth of cells on the pore walls. We exploit the slenderness of a pore that is typical in such a scenario, to derive a reduced model that enables a comprehensive analysis of the system to be performed. We derive analytical solutions in a particular case of a nearly piecewise constant growth law and compare these with numerical solutions of the reduced model. Qualitative comparisons of tissue morphologies predicted by our model, with those observed experimentally, are also made. We demonstrate how the simplified system may be used to make predictions on the design of a tissue-engineering scaffold and the appropriate operating regime that ensures a desired level of tissue growth.