Carol J. Belfiore

Steroidogenic enzyme activity after acute activation of protein kinase (PK) A and PKC in ovine small and large luteal cells.

Intracellular effector systems which utilize PKA and PKC can be pharmacologically activated by forskolin and phorbol 12-myristate 13-acetate (PMA) and appear to be important for regulation of steroidogenesis by cells of the corpus luteum. In this study the effect of pharmacologic activation of PKA (forskolin) or PKC (PMA) on the activity of adenylate cyclase, cholesterol esterase, P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5, delta 4 isomerase (3 beta HSD) was determined. Basal adenylate cyclase activity (as measured by intracellular and secreted cAMP) was extremely low in both large and small luteal cells. Forskolin stimulated adenylate cyclase activity in both large and small luteal cells but progesterone production was increased only in small cells. PMA inhibited progesterone production by large and forskolin-stimulated small cells without altering adenylate cyclase activity. Basal cholesterol esterase activity was greater in small than in large cells and was stimulated by forskolin only in small cells. PMA did not significantly alter cholesterol esterase activity in either cell type. Activity of P450scc or 3 beta HSD was measured by conversion of hydroxylated cholesterol derivatives (P450scc) or pregnenolone (3 beta HSD) to progesterone. Although basal progesterone production was 47 times greater in large than small cells, there was only 5.1 (P450scc) and 6.4 (3 beta HSD) times greater enzyme activity in large than in small luteal cells. Activation of PKA and/or PKC did not alter the activity of P450scc or 3 beta HSD in either cell type.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cytochrome P450scc synthesis and activity in the ovine corpus luteum

The rate-limiting step in luteal biosynthesis of progesterone consists of cleavage of the side chain of cholesterol by mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc) to form pregnenolone. Luteal mRNA encoding P450scc, quantitated on selected days of the 16-day ovine estrous cycle, was similar on days 3 and 6, increased by 2-fold on day 9 (P < 0.05) and remained elevated on day 15. Levels of P450scc mRNA on day 15 of pregnancy were not different from those found on any day of the cycle (P < 0.05). To determine whether levels of mRNA encoding P450scc are hormonally regulated, ewes on day 10 of the estrous cycle were injected with hCG or prostaglandin F2 alpha (PGF2 alpha). P450scc mRNA was not increased for up to 36 h after injection of hCG, nor decreased within 8 h after injection of PGF2 alpha (P < 0.05). An assay for P450scc activity was developed which utilized ovine small and large luteal cells in the presence of 22R-hydroxycholesterol and ovine high density lipoprotein. Enzyme activity was quantitated by measurement of progesterone production. In small luteal cells activation of the protein kinase A (PKA) second-messenger system by treatment with LH resulted in 910% increase in progesterone production without altering activity of P450scc. Activation of the protein kinase C (PKC) second-messenger system with phorbol 12-myristate 13-acetate caused a 51% reduction in progesterone secretion from large luteal cells but did not alter activity of P450scc. These findings suggest that in mature luteal tissue steady state levels of mRNA encoding P450scc, and enzyme activity are independent of acute regulation by activation of PKA or PKC second-messenger systems.

Stress-sensitive nutrient consumption via steady and non-reversing dynamic shear in continuous-flow rotational bioreactors ☆

Stress-sensitive biological response is simulated in a modified parallel-disk viscometer that implements steady and unidirectional dynamic shear under physiological conditions. Anchorage-dependent mammalian cells adhere to a protein coating on the surface of the rotating plate, receiving nutrients and oxygen from an aqueous medium that flows radially and tangentially, accompanied by transverse diffusion in the z-direction toward the active surface. This process is modeled as radial convection and axial diffusion with angular symmetry in cylindrical coordinates. The reaction/diffusion boundary condition on the surface of the rotating plate includes position-dependent stress-sensitive nutrient consumption via the zr- and zTheta-elements of the velocity gradient tensor at the cell/aqueous-medium interface. Linear transport laws in chemically reactive systems that obey Curies theorem predict the existence of cross-phenomena between scalar reaction rates and the magnitude of the second-rank velocity gradient tensor, selecting only those elements of nabla v experienced by anchorage-dependent cells that are bound to protein-active sites. Stress sensitivity via the formalism of irreversible thermodynamics introduces a zeroth-order contribution to heterogeneous reaction rates that must be quenched when nutrients, oxygen, chemically anchored cells, or vacant active protein sites are not present on the surface of the rotating plate. Computer simulations of nutrient consumption profiles via simple nth-order kinetics (i.e., n=1,2) suggest that rotational bioreactor designs should consider stress-sensitivity when the shear-rate-based Damköhler number (i.e., ratio of the stress-dependent zeroth-order rate of nutrient consumption relative to the rate of nutrient diffusion toward active cells adhered to the rotating plate) is greater than approximately 25% of the stress-free Damköhler number. Rotational bioreactor simulations are presented for simple 1st-order, simple 2nd-order, and complex stress-free kinetics, where the latter includes a 4th-order rate expression that considers adsorption/desorption equilibria via the Fowler-Guggenheim modification of the Langmuir isotherm for receptor-mediated cell-protein binding, accompanied by the formation of receptor complexes. Dimensionless parameters are identified to obtain equivalent stress-free nutrient consumption in the exit streams of 2-dimensional creeping-flow rotational bioreactors and 1-dimensional laminar-flow tubular bioreactors. Modulated rotation of the active plate at physiological frequencies mimics pulsatile cardiovascular flow and demonstrates that these rotational bioreactors must operate above the critical stress-sensitive Damköhler number, identified under steady shear conditions, before dynamic shear has a distinguishable effect on bioreactor performance.

Tubular bioreactor models that include Onsager–Curie scalar cross-phenomena to describe stress-dependent rates of cell proliferation

The theory of heterogeneous catalysis in chemical reactors is employed to simulate laminar flow through tubes at large mass transfer Peclet numbers in which anchorage-dependent cells (i) adhere to a protein coating on the inner surface at r=R(wall), (ii) receive nutrients and oxygen from an aqueous medium via transverse diffusion toward the active wall, and (iii) proliferate in the presence of viscous shear at the cell/aqueous-medium interface. This process is modeled as convective diffusion in cylindrical coordinates with chemical reaction at the boundary, where chemical reaction describes the rate of nutrient consumption. The formalism of irreversible thermodynamics is employed to describe an unusual coupling between viscous shear, or velocity gradients at the cell/aqueous-medium interface, and rates of nutrient consumption. Linear transport laws in chemically reactive systems that obey Curies theorem predict the existence of cross-phenomena between fluxes (i.e., scalar reaction rates) and driving forces (i.e., 2nd-rank velocity gradient tensor) whose tensorial ranks differ by an even integer-in this case, two. This methodology for stress-dependent chemical reactions yields an additional zeroth-order contribution, via the magnitude of the velocity gradient tensor, to heterogeneous kinetic rate expressions because nutrient consumption and cell proliferation are stress-sensitive. Computer simulations of nutrient consumption suggest that bioreactor designs should consider stress-sensitive reactions when the shear-rate-based Damköhler number (i.e., defined for the first time in this study as the stress-dependent zeroth-order rate of nutrient consumption relative to the rate of nutrient diffusion toward active cells adhered to the tube wall) is greater than 10-20% of the stress-free Damköhler number. Models of bioreactor performance are presented for simple 1st-order, simple 2nd-order, and complex chemical kinetic rate expressions, where the latter considers adsorption/desorption equilibria via the Fowler-Guggenheim modification of the Langmuir isotherm for cell-protein docking on active sites, accompanied by cell-cell attraction. Stress sensitivity is magnified in physically realistic cell-based tubular bioreactors with complex stress-free kinetic rate expressions relative to simulations with simple 1st- and 2nd-order kinetics.

Stress-Sensitive Tissue Regeneration in Viscoelastic Biomaterials Subjected to Modulated Tensile Strain

This research contribution addresses the mechanochemistry of intra-tissue mass transfer for nutrients, oxygen, growth factors, and other essential ingredients that anchorage-dependent cells require for successful proliferation on biocompatible surfaces. The unsteady state reaction-diffusion equation (i.e., modified diffusion equation) is solved according to the von Kármán-Pohlhausen integral method of boundary layer analysis when nutrient consumption and tissue regeneration are stimulated by harmonically imposed stress. The mass balance with diffusion and stress-sensitive kinetics represents a rare example where the Damköhler and Deborah numbers appear together in an effort to simulate the development of mass transfer boundary layers in porous viscoelastic biomaterials. The Boltzmann superposition integral is employed to calculate time-dependent strain in terms of the real and imaginary components of dynamic compliance for viscoelastic solids that transmit harmonic excitation to anchorage-dependent cells. Rates of nutrient consumption under stress-free conditions are described by third-order kinetics which include local mass densities of nutrients, oxygen, and attached cells that maintain dynamic equilibrium with active protein sites in the porous matrix. Thinner nutrient mass transfer boundary layers are stabilized at shorter dimensionless diffusion times when the stress-free intra-tissue Damköhler number increases above its initial-condition-sensitive critical value. The critical stress-sensitive intra-tissue Damköhler number, above which it is necessary to consider the effect of harmonic strain on nutrient consumption and tissue regeneration, is proportional to the Deborah number and corresponds to a larger fraction of the stress-free intra-tissue Damköhler number in rigid biomaterials.

Nutrient diffusion and simple nth-order consumption in regenerative tissue and biocatalytic sensors.

This contribution addresses intra-tissue molar density profiles for nutrients, oxygen, growth factors, and other essential ingredients that anchorage-dependent cells require for successful proliferation on biocompatible surfaces. One-dimensional transient and steady state models of the reaction-diffusion equation are solved to correct a few deficiencies in the first illustrative example of diffusion and zeroth-order rates of consumption in tissues with rectangular geometry, as discussed in Ref. [(Griffith and Swartz, 2006) 1]. The functional form of the molar density profile for each species depends on geometry and the magnitude of the species-specific intra-tissue Damköhler number. The tissues central core is reactant starved at high consumption rates and low rates of intra-tissue diffusion when the Damköhler number exceeds its geometry-sensitive critical value. Ideal tissue engineering designs avoid the diffusion-limited regime such that attached cells are exposed to all of the ingredients required for proliferation everywhere within a regenerative matrix. Analytical and numerical molar density profiles that satisfy the unsteady state modified diffusion equation with pseudo-homogeneous n(th)-order rates of intra-tissue consumption (i.e., n=0,1,2) allow one to (i) predict von Kármán-Pohlhausen mass transfer boundary layer thicknesses, measured inward from the external biomaterial surface toward its central core, and, most importantly, (ii) estimate the time required to achieve steady state conditions for regenerative tissue growth and biocatalytic sensing.

Electric-field-enhanced nutrient consumption in dielectric biomaterials that contain anchorage-dependent cells

This research contribution addresses electric-field stimulation of intra-tissue mass transfer and cell proliferation in viscoelastic biomaterials. The unsteady state reaction-diffusion equation is solved according to the von Kármán-Pohlhausen integral method of boundary layer analysis when nutrient consumption and tissue regeneration occur in response to harmonic electric potential differences across a parallel-plate capacitor in a dielectric-sandwich configuration. The partial differential mass balance with diffusion and electro-kinetic consumption contains the Damköhler (Λ(2)) and Deborah (De) numbers. Zero-field and electric-field-sensitive Damköhler numbers affect nutrient boundary layer growth. Diagonal elements of the 2nd-rank diffusion tensor are enhanced in the presence of weak electric fields, in agreement with the formalism of equilibrium and nonequilibrium thermodynamics. Induced dipole polarization density within viscoelastic biomaterials is calculated via the real and imaginary components of the complex dielectric constant, according to the Debye equation, to quantify electro-kinetic stimulation. Rates of nutrient consumption under zero-field conditions are described by third-order kinetics that include local mass densities of nutrients, oxygen, and attached cells. Thinner nutrient boundary layers are stabilized at shorter dimensionless diffusion times when the zero-field intra-tissue Damköhler number increases above its initial-condition-sensitive critical value [i.e., {Λ(2)(zero-field)}(critical)≥53, see Eq. (23)], such that the biomaterial core is starved of essential ingredients required for successful proliferation. When tissue regeneration occurs above the critical electric-field-sensitive intra-tissue Damköhler number, the electro-kinetic contribution to nutrient consumption cannot be neglected. The critical electric-field-sensitive intra-tissue Damköhler number is proportional to the Deborah number.

Dynamic shear-stress-enhanced rates of nutrient consumption in gasliquid semi-continuous-flow suspensions

Abstract The primary objective of this investigation is to establish guidelines for generating significant mammalian cell density in suspension bioreactors when stress-sensitive kinetics enhance the rate of nutrient consumption. Ultra-low-frequency dynamic modulations of the impeller (i.e., 35104 Hz) introduce time-dependent oscillatory shear into this transient analysis of cell proliferation under semi-continuous creeping flow conditions. Greater nutrient consumption is predicted when the amplitude A of modulated impeller rotation increases, and stress-kinetic contributions to nutrient consumption rates increase linearly at higher modulation frequency via an application of fluctuation-dissipation response. Interphase mass transfer is required to replace dissolved oxygen as it is consumed by aerobic nutrient consumption in the liquid phase. The theory and predictions described herein could be important at small length scales in the creeping flow regime where viscous shear is significant at the interface between the nutrient medium and isolated cells in suspension. Two-dimensional flow around spherically shaped mammalian cells, suspended in a Newtonian culture medium, is analyzed to calculate the surface-averaged magnitude of the velocity gradient tensor and modify homogeneous rates of nutrient consumption that are stimulated by viscous shear, via the formalism of stress-kinetic reciprocal relations that obey Curies theorem in non-equilibrium thermodynamics. Time constants for stress-free free and stress-sensitive stress nutrient consumption are defined and quantified to identify the threshold (i.e., stress,threshold) below which the effect of stress cannot be neglected in accurate predictions of bioreactor performance. Parametric studies reveal that the threshold time constant for stress-sensitive nutrient consumption stress,threshold decreases when the time constant for stress-free nutrient consumption free is shorter. Hence, stress,threshold depends directly on free. In other words, the threshold rate of stress-sensitive nutrient consumption is higher when the stress-free rate of nutrient consumption increases. Modulated rotation of the impeller, superimposed on steady shear, increases stress,threshold when free is constant, and stress,threshold depends directly on the amplitude A of these angular velocity modulations.