Osteogenic preconditioning in perfusion bioreactors improves vascularization and bone formation by human bone marrow aspirates.

Cell-derived extracellular matrix (ECM) provides a niche to promote osteogenic differentiation, cell adhesion, survival, and trophic factor secretion. To determine whether osteogenic preconditioning would improve the bone-forming potential of unfractionated bone marrow aspirate (BMA), we perfused cells on ECM-coated scaffolds to generate naïve and preconditioned constructs, respectively. The composition of cells selected from BMA was distinct on each scaffold. Naïve constructs exhibited robust proangiogenic potential in vitro, while preconditioned scaffolds contained more mesenchymal stem/stromal cells (MSCs) and endothelial cells (ECs) and exhibited an osteogenic phenotype. Upon implantation into an orthotopic calvarial defect, BMA-derived ECs were present in vessels in preconditioned implants, resulting in robust perfusion and greater vessel density over the first 14 days compared to naïve implants. After 10 weeks, human ECs and differentiated MSCs were detected in de novo tissues derived from naïve and preconditioned scaffolds. These results demonstrate that bioreactor-based preconditioning augments the bone-forming potential of BMA.

Nondestructive fluorescence lifetime imaging and time-resolved fluorescence spectroscopy detect cartilage matrix depletion and correlate with mechanical properties.

Tissue engineers utilize a battery of expensive, time-consuming and destructive techniques to assess the composition and function of engineered tissues. A nondestructive solution to monitor tissue maturation would reduce costs and accelerate product development. As a first step toward this goal, two nondestructive, label-free optical techniques, namely multispectral fluorescent lifetime imaging (FLIm) and time-resolved fluorescence spectroscopy (TRFS), were investigated for their potential in evaluating the biochemical and mechanical properties of articular cartilage. Enzymatic treatments were utilized to selectively deplete cartilage of either collagen or proteoglycan, to produce a range of matrix compositions. Samples were assessed for their optical properties using a fiber-coupled optical system combining FLIm and TRFS, their biochemical and mechanical properties and by histological staining. Single and multivariable correlations were performed to evaluate relationships among these properties. FLIm- and TRFS-derived measurements are sensitive to changes in cartilage matrix and correlate with mechanical and biochemical assays. Mean fluorescence lifetime values extracted from FLIm images (375-410 nm spectral band) showed strong, specific correlations with collagen content (R2 = 0.79, p < 0.001) and tensile properties (R2 = 0.45, p = 0.02). TRFS lifetime measurements centered at 520 nm (with a 5 nm bandwidth) possessed strong, specific correlations with proteoglycan content (R2 = 0.59, p = 0.001) and compressive properties (R2 = 0.71, p < 0.001). Nondestructive optical assessment of articular cartilage, using a combination of FLIm- and TRFS-derived parameters, provided a quantitative method for determining tissue biochemical composition and mechanical function. These tools hold great potential for research, industrial and clinical settings.

Effect of encapsulation on plasminogen activator delivery to the microcirculation and its implications for bleeding.

BACKGROUND AND PURPOSE: It is known that encapsulation can alter the delivery of plasminogen activators by flow to accelerate fibrinolysis while other experimental studies suggest encapsulation may reduce the risk of hemorrhage with administration of the agent. The aim of this research is to resolve the effect of encapsulation on fibrinolysis and bleeding in the microcirculation. METHODS: An established rabbit model of fibrinolytic hemorrhage was utilized to explore the potential of encapsulation to limit bleeding. Equal dosages of free or microencapsulated streptokinase (MESK) were infused to initiate thrombolysis of small vessel clots while tracking blood loss. RESULTS: Compared to free streptokinase, significant improvements in bleeding were observed with MESK as demonstrated by (1) delayed onset of bleeding, (2) shortened duration, and (3) reduction in the volume of lost blood, consistent with less systemic fibrinogen degradation. CONCLUSIONS: Findings demonstrate that encapsulation of streptokinase can inhibit clot lysis in small vessels. Combined with prior work on accelerated thrombolysis, results suggest a time-based regimen for avoiding bleeding complications during thrombolytic therapy with encapsulated agent.

Supplementation of fibrin gels with sodium chloride enhances physical properties and ensuing osteogenic response.

Modifying the relative concentrations of fibrinogen and thrombin can control the physical properties of fibrin gels, while the viability of associated cells has been linked to the gel's final network structure. It was hypothesized that increasing the gel ionic strength during fabrication through supplementation with sodium chloride (NaCl) would provide an improved approach for tailoring the physical properties of fibrin gels and maintaining the viability and osteogenic potential of entrapped cells. Fibrin gels were formed by mixing fibrinogen, thrombin and calcium chloride with varying masses of NaCl (0-4.40% w/v), and the osteogenic potential of entrapped human mesenchymal stem cells (MSC) was examined over 14 days. Physical properties including gelation time, compressive modulus and fiber diameter were dependent upon NaCl content, with gels containing 2.60% NaCl possessing compressive moduli threefold higher than gels without NaCl. Alkaline phosphatase activity was highest for MSC entrapped in gels containing 2.15-2.60% NaCl after 14 days, and all gels exhibited increased calcium incorporation over the culture period. These data confirm that varying the salt concentration of the pre-gel solution can modulate the material properties of fibrin constructs without additional fibrinogen or thrombin, thereby offering a new approach for generating improved cell transplantation vehicles for use in bone tissue regeneration.

A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction.

In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

Distributed intraclot thrombolysis: mechanism of accelerated thrombolysis with encapsulated plasminogen activators.

BACKGROUND: The delivery of encapsulated plasminogen activators has demonstrated enhanced thrombolysis in vivo in several models. The mechanism of such improvement has not previously been established. OBJECTIVES: We explored in vitro the mechanism by which microencapsulation of streptokinase in polymeric microparticles accelerates clot digestion and reduces reperfusion times by as much as an order of magnitude in vivo. METHODS: The efficacy of microencapsulated streptokinase (MESK) was directly compared with identical dosages of unencapsulated streptokinase (FREE SK) at three initial pressure drops using clots formed of plasma or whole blood in 0.2-cm inner diameter glass capillary tubes. RESULTS: MESK demonstrated accelerated flow restoration compared with FREE SK for each condition in plasma (23.8 +/- 4.5% faster) and whole blood clots (17.2 +/- 9.2% faster). Images collected by light microscopy show sites of thrombolysis internal to the clot only with MESK while the spatial distribution of fluorescently labeled streptokinase by confocal microscopy confirms greater penetration of the encapsulated agent compared with unencapsulated streptokinase. Digestion thus proceeds in three dimensions rather than restricted to a two-dimensional lysis front. CONCLUSIONS: The improved clot penetration with MESK establishes enhanced transport with encapsulation and the concept of distributed intraclot thrombolysis as a basis for the accelerated dissolution observed with encapsulated plasminogen activators in vivo.

Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen.

Transient generation of reactive oxygen or nitrogen (ROS/RNS), detected with dihydrodichlorofluoroscein by fluorescence microscopy, occurs within minutes of exposing cells to ionizing radiation. In the 1-10 Gy dose range, the amount of ROS/RNS produced/cell is constant, but the percentage of producing cells increases with dose (20 to 80%). Reversible depolarization of the mitochondrial membrane potential () and decrease in fluorescence of a mitochondria-entrapped dye, calcein, are observed coincidentally. Radiation-induced ROS/RNS, depolarization, and calcein fluorescence decrease are inhibited by the mitochondrial permeability transition inhibitor, cyclosporin A, but not the structural analogue, cyclosporin H. Radiation-stimulated ROS/RNS is also inhibited by overexpressing the Ca(2+)-binding protein, calbindin 28K, or treating cells with an intracellular Ca(2+) chelator. Radiation-induced ROS/RNS is observed in several cell types with the exception of rho(o) cells deficient in mitochondrial electron transport. rho(o) cells show neither radiation-induced ROS/RNS production nor depolarization. We propose that radiation damage in a few mitochondria is transmitted via a reversible, Ca(2+)-dependent mitochondrial permeability transition to adjacent mitochondria with resulting enhanced ROS/RNS generation. Measurements of radiation-induced mitogen-activated protein kinase activity indicate that this sensing/amplification mechanism is necessary for activation of some cytoplasmic signaling pathways by low doses of radiation.

Shortening deactivation of cardiac muscle: physiological mechanisms and clinical implications.

UNLABELLED: PHYSIOLOGICAL MECHANISM: A rapid change of length applied during isometric contraction of skeletal or cardiac muscle may result in redeveloped tension less than appropriate for the new length because of "deactivation" of the contractile system. The amount of shortening deactivation is directly related to both the time during the contraction when the length change occurs and to the extent of muscle shortening. If the muscle is permitted to shorten early in the contraction, the redeveloped tension will be appropriate to the new length as predicted from the classic Frank-Starling relationship. However, the same length change, which is imposed later in the contraction, results in a redeveloped tension that is less than predicted. Furthermore, a greater change in length results in less tension being redeveloped than if a smaller length decrement is applied at the same time during the contraction. It has been demonstrated that the reduced tension during active muscle shortening is associated with reduced affinity of troponin C for Ca2+. The free Ca2+ is then picked up by the SR, with less Ca2+ available for tension development until the subsequent contraction. CLINICAL SIGNIFICANCE: Although the clinical significance of shortening deactivation remains speculative, it seems likely that in the intact heart deactivation would affect myocardial O2 consumption. The decreased efficiency with which the heart maintains a given stroke work against a high afterload might be related to the lesser degree of fiber shortening and, therefore, less shortening deactivation. Conversely, it is well-known that the same level of stroke work accomplished by an increase in end-diastolic volume requires much less O2. This may be related, at least in part, to the greater degree of shortening with an accompanying increase in deactivation under the latter conditions. For example, in congestive heart failure where ejection fraction and fiber shortening are minimal, the maintenance of the longer fiber lengths could significantly increase the MVO2. Ford has suggested that the deactivating effect of shortening produced by afterload reduction would limit energy expenditure, therefore, exerting a favorable effect on the failing myocardium. It would also seem that an inotropic agent that increased shortening deactivation might compensate for the increased MVO2 caused by the inotrope and have a favorable effect on cardiac work. From most of the studies we have reviewed, it appears likely that shortening deactivation acts as a physiological "feedback" mechanism that affects afterload and in turn, myocardial oxygen consumption. Pathological situations such as acidosis and ischemia have been associated with reduced myofilament Ca2+ sensitivity or affinity and depressed cardiac contractility. Is it then possible that interventions that increase Ca2+ sensitivity might favorably alter ventricular pressure-volume relations during ejection and improve myocardial function by reducing the magnitude of shortening deactivation? Whatever the mechanism and clinical significance, future investigations will help to define the role of shortening deactivation in modifying ventricular function.

Depression of papillary muscle contractility by plasma incubated with pneumococci.

Mechanisms of death in pneumococcal disease are poorly understood. We have previously shown that intravenous pneumococcal products in dogs caused a mean decrease in cardiac output of 58%. The present study used measurements of the force and rate of contraction of isolated rabbit papillary muscle to determine whether pneumococci (PNC) altered myocardial contractility. Nine papillary muscles were superfused with various solutions including Tyrode's, Tyrode's incubated with sonicated type 1 PNC, normal rabbit plasma, and rabbit plasma incubated with PNC. Compared to untreated Tyrode's solution, PNC-treated Tyrode's solution did not alter papillary muscle contractility. However, compared to untreated rabbit plasma, plasma incubated with PNC caused a mean decrease of 18% in the force of contraction and 16.7% in the maximum rate of force development in nine studies. We conclude that PNC do not directly affect papillary muscle contractility. However, the interaction of PNC and plasma does cause a decrease in rabbit papillary muscle contractility.

Effects of active shortening on tension development of rabbit papillary muscle.

Duration and intensity of force development have been shown to be less during active muscle shortening than during isometric contraction. The purpose of this study was to compare force developed during controlled shortening with that predicted by the Frank-Starling relation. Paillary muscles from the right ventricles of rabbits were arranged for isometric tension recording, and isometric contractions were recorded at several lengths. The muscles were then permitted to shorten at velocities of 0.2--6 mm/s, shortening beginning 150--200 ms after the stimulus. Length-tension-time curves constructed from the isometric contractions were used to determine predicted shortening tension (Pp), which was compared with actual tension during shortening (Ps) at corresponding times and lengths. Ps was significantly less than Pp and the ratio Ps/Pp decreased with increasing velocity of shortening. The decrease in Ps/Pp was directly related to the duration of shortening (P less than 0.001), suggesting that the fall of shortening tension reflected both the Hill force-velocity relation and shortening deactivation.

The effect of nitroglycerin on gas exchange, hemodynamics, and oxygen transport in patients with chronic obstructive pulmonary disease.

The present study was designed to investigate pharmacological vasodilation in patients with chronic obstructive pulmonary disease (COPD). This may have long-term therapeutic implications. Sublingual nitroglycerin (GTN) was administered to 16 patients with COPD and no evidence of organic heart disease (except one patient) in an attempt to determine severity of the decrease in arterial oxygenation. Gas exchange and hemodynamic studies were performed before and after GTN. Alverolar-arterial O2 tension gradient increased 5 mm Hg, arterial PO2 decreased 2 mm Hg, and cardiac output decreased from 4.36 to 3.85 liters/min. There was a decrease in pulmonary artery pressures, but pulmonary vascular resistance did not change. Total O2 transport (the product of cardiac output and arterial O2 content) decreased, due mainly to a decrease in cardiac output. From these studies we conclude that GTN significantly decreases O2 transport in patients with COPD and normal left ventricular function; this decrease is due mainly to a reduction in cardiac output. Impairment of gas exchange is slight. Based on these considerations pharmacological vasodilation with nitroglycerine in COPD is probably not warranted.

Biological activity of dilute isoproterenol solution stored for long periods in plastic bags.

The effect of long-term storage in polyvinyl chloride bags on the biological activity and the chemical stability of isoproterenol was studied. A freshly prepared solution of isoproterenol, isoproterenol solution stored for six months and isoproterenol stored for 30 months were infused into the coronary circulation of 14 isolated rabbit hearts; heart rates were determined. Spectrophotometric studies to determine stability at various pH levels were also done. Aging of the solutions at pH 4 and at a concentration of 4 microgram/ml caused no significant impairment of their potency. Solutions above pH 6 were unstable.

Cardiomyopathy in the Southwest American Indian.

Six Southwestern American Indian patients are reported to have primary myocardial disease or congestive failure of unknown cause. Two are Navajo, three are Laguna, and one is Isleta. Clinical and laboratory features are discussed. This disease appears in culturally traditional Indians and is not associated with contact with American civilization. It occurs in probably adequately nourished Indians in the majority of whom excessive alcohol consumption is not found. No familial cases were noted. This syndrome is probably not uncommon in the American Indian.