New acrylic microspheres for arterial embolization: combining radiopacity for precise localization with immobilized thrombin to trigger local blood coagulation

Particles currently used in arterial embolization therapy have several disadvantages, most importantly their radiolucency. This means the radiologist cannot precisely asses the fate of embolization particles. Microspheres that combine two additional features have been designed. By incorporating an iodine containing monomer, radiopaque microspheres were obtained that display good visibility under standard X-ray conditions. Incorporation of methacrylic acid makes the surface of the spheres suitable for surface functionalization. Here, thrombin was covalently attached to the surface of the radiopaque microspheres. By induction of a thrombus, improved anchoring of the embolization spheres in the blood vessel can be obtained. The immobilized thrombin induced a biphasic response of the blood namely: (1) fast deposition of fibrin on the surface resulting in sphere aggregation and (2) additional thrombin generation in the surrounding blood and a subsequent local thrombus formation. These microspheres with both intrinsic X-ray visibility and a biofunctionalized surface can potentially improve embolization therapies.     

Ultraviolet light-induced modification of crosslinked hyaluronan gels

Hyaluronan (HA) gels (hylans) crosslinked with divinyl sulfone (DVS) are highly biocompatible and can be structurally modified to obtain desired mechanical properties that are attractive for their use as tissue-engineering scaffolds. However, unmodified hylan gels are not good substrates for cell attachment or infiltration, likely as a result of their smooth surface and the highly anionic nature of HA. This study investigated whether the cell-adhering characteristics of hylan gels could be enhanced by irradiation with ultraviolet (UV) light, with or without prior dehydration. The attachment and proliferation of neonatal rat smooth muscle cells atop these gels was compared with that on unmodified (control; C) or dehydrated (D) gels. UV-induced changes to gel structure and chemistry were characterized by confocal and electron microscopy, and fluorphore-assisted carbohydrate electrophoresis (FACE). Cell attachment was sparse on both unmodified (C) and dehydrated (D) gels. Significantly higher levels of cell attachment were observed on the surface of irradiated (UV) and dehydrated-irradiated (DUV) gels, likely because of texturing of the gel surface by UV light. In addition, dehydration of gels before UV irradiation created irregular pore-like structures through which cells appeared to migrate into the interior. FACE assays demonstrated that UV-irradiation alters the chemistry of HA, causing limited breakdown of HA chains and DVS crosslinks within gel and possibly creating new crosslinks that have not yet been identified. Because the hylan gels are altered structurally and chemically, binding of cells to the material is likely to be more permanent than possible by other approaches, such as coating of cell-adhesive matrix factors on the gel surface, described previously. The significance of this work is that we have developed a technique for the modification of DVS-crosslinked HA (hylans) to enhance their performance as a cellular scaffold for tissue-engineering applications.     

Mechanism of the congestion of lymph nodes induced by ellagic acid in rats

Intravenous injection of ellagic acid (EA, 30 mg/Kg), an activator of the Hageman factor, induced congestion of lymph nodes and dilatation of the spleen in rats. The dilatation of the spleen was inhibited by heparin, thrombin, defibrase, clocoumarol. lambda carrageenan, SBTI, PCR 4099 and CCI 17810. The congestion of lymph nodes was inhibited by heparin, thrombin, defibrase, clocoumarol, SBTI, lambda carrageenan, aspirin, indomethacin and ketoprofen, phentolamine and hexamethonium. Thrombin, defibrase, lambda carrageenan did not induce any congestion of the lymphoid tissues. These results suggest that the dilatation of the spleen induced by ellagic acid would result from blood coagulation, platelet stimulation and kinin formation. The congestion of lymph nodes would depend on kinins, blood coagulation and prostaglandins. The activation of Hageman factor in acute inflammatory reactions could mediate the early congestion of lymph nodes.     

Fibrin gel formation in a shear flow.

Blood clots are made up of platelets and fibrin gel, and the relative amount of fibrin is strongly influenced by the shear rate. In order to explore this phenomenon, this paper presents a model of fibrin gel formation over the surface of an injured blood vessel in a shear flow. A condition for gelation including source and sink terms of polymer is derived. A simplified model of coagulation, involving activation and inhibition of the enzyme thrombin and thrombin-mediated production of fibrin monomer, is combined with the model of gelation to explore how the shear rate and other parameters control the formation of fibrin gel. The results show that the thrombin inhibition rate, the gel permeability and the shear rate are key parameters in regulating the height of the fibrin gel.     

Cardiac sympathetic adrenergic pathways in which synaptic transmission is blocked by atropine sulphate

1. Stimulation of the thoracic sympathetic nerve trunk caused a rise in arterial blood pressure and heart rate which was reduced but not abolished during the infusion of hexamethonium chloride, pentolinium tartrate, mecamylamine or tetraethylammonium chloride.

2. Preganglionic stimulation no longer elicited a synchronous sC elevation in the post-ganglionic nerves. The persistent blood pressure and heart rate response was associated with an asynchronous discharge in these nerves.

3. Both the persistent response and the asynchronous discharge were abolished by the intravenous injection of atropine sulphate in doses of 30 μg/kg.

4. Atropine had no effect on spike amplitude or shape, conduction velocity or frequency response in either the pre- or post-ganglionic nerve trunks. Nor did it affect the rise in blood pressure and heart rate evoked by stimulation of the post-ganglionic cardiac sympathetic nerves.

5. It is concluded that atropine partially blocked synaptic transmission in cardiac sympathetic adrenergic pathways.

     

Neuroeffector mechanisms of the defense reaction in the rat

Electrical stimulation of the dorsal periaqueductal gray matter (DPAG) eliciting flight behavior in awake rats caused an increase in arterial blood pressure (BP), heart rate (HR) and respiration in rats anesthetized with urethane. The hypertension was markedly reduced by 5 mg/kg of intravenously injected hexamethonium or bretylium, virtually abolished by 5 mg/kg of phentolamine and partially antagonized by 0.1 mg/kg of the α₁-adrenoceptor blocker, prazosin. The tachycardia induced by DPAG stimulation was partially antagonized by hexamethonium or bretylium and abolished by propranolol (5 mg/kg, IV) or practolol (5 mg/kg, IV), but not affected by N-butylscopolamine (10 mg/kg, IV). Phentolamine increased basal HR and abolished the tachycardic response caused by either brain stimulation or intravenous noradrenaline. Prazosin moderately decreased the response to noradrenaline, but did not affect basal HR or the tachycardia induced by brain stimulation. The increase in respiratory amplitude occurring during brain stimulation was abolished by phentolamine as well as by prazosin, while the increase in respiratory rate was moderately reduced by phentolamine and propranolol. These results demonstrate that the cardiovascular component of the defense reaction of the rat is almost entirely due to a sharp increase in sympathetic tone. They also suggest that the hyperventilation induced by aversive brain stimulation is modulated by central and peripheral adrenergic mechanisms.     

Regional distribution of vasoconstrictor tone in acute spinal rats

Rats, chronically instrumented with an electromagnetic flow probe around the carotid, superior mesenteric, or renal artery, or the terminal aorta as well as having an arterial and venous catheter, were anesthetized with either and submitted to high spinal cord transection. One hour later, when the rats had recovered consciousness and the arterial pressure had recovered partially, hexamethonium was intravenously injected for ganglionic blockade. Peripheral resistance (arterial pressure/regional flow) was decreased significantly by ganglionic blockade in the carotid and renal areas but not in the superior mesenteric and hindquarter (terminal aortic) areas. This suggests the presence of sizable vasoconstrictor tone to resistance vessels in the carotid and renal areas but not in the superior mesenteric and hindquarter areas in the acute spinal rat. This distribution of vasoconstrictor tone is similar to that in intact conscious rats in the resting state and suggests the possibility that the vasoconstrictor tone for resistance vessels in intact rats is also for the most part generated in the spinal cord. Spinal transection decreased blood flow in all the four regions, suggesting a decrease in cardiac output due to dilation of capacitance vessels. It is possible that, in intact rats at rest, the so-called medullary vasomotor center is sending tonic impulses for the most part to capacitance vessels.     

Controlled preparation of thin fibrin films immobilized at solid surfaces

A technique for coating surfaces with attached fibrin structures without the formation of fibrin gel in bulk solution was developed. It is based on the catalytic effect of the surface-bound thrombin on fibrinogen stabilized with inhibitor which inhibits thrombin in solution but not the thrombin on the surface. Such an inhibitor is antithrombin, the effect of which may be enhanced with heparin. Fibrinogen is first adsorbed on the substrate surface and then incubated with thrombin. The unbound thrombin is washed out and the surface is incubated with fibrinogen solution containing antithrombin III and heparin. A fibrin gel forms at the surface by the action of surface-bound thrombin on ambient fibrinogen solution; however, the gel formation in bulk solution catalyzed by thrombin partially released from the surface is suppressed. By utilizing antithrombin-independent inhibitors or repeating thrombin binding and incubation with fibrinogen solution, the amount of surface-attached fibrin gel can be controlled. The formation of immobilized fibrin networks was observed using surface plasmon resonance and turbidity measurements and morphology was observed by TEM, SEM, and AFM. Using this technique, a porous scaffold made of polylactide fibers was coated with fibrin without filling the space between fibers with a bulk fibrin gel. The technique makes it possible to coat the inner surface of porous scaffolds with surface-attached fibrin gel while preserving free volume for cell migration into the pores.     

Reduction of sensory responses to passive movements of inflamed knee joints by hylan, a hyaluronan derivative

 Hyaluronan (sodium hyaluronate) is a glycosaminoglycan that is present in all joint tissues. Painful arthritic joints have been characterized by hyaluronan of reduced elastoviscosity. The purpose of this investigation was to determine whether hyaluronan has an influence on joint nociceptor sensitivity and whether restoration of elastoviscosity would decrease nerve responses from nociceptive afferent fibers in arthritic joints. Nerve impulse activity was recorded from nociceptive afferent fibers of the medial articular nerve in anesthetized cats. An acute experimental arthritis was produced by intra-articular injection of kaolin and carrageenan. This caused, within 3 h, the development of ongoing nerve activity and enhancement of nerve impulse responses to passive movements in the normal range of the joint. Intra-articular injection of an elastoviscous solution of hylan, a hyaluronan derivative, significantly reduced both the ongoing activity and the movement-evoked responses in 1–2 h. This effect was not obtained when a nonelastoviscous solution of hylan was injected into the inflamed joint. The results indicate that intra-articularly injected elastoviscous solutions of hylan reduced nociceptive activity in inflamed joints through an elastoviscous, rheological effect on nociceptive afferent fibers through the intercellular matrix in which these fibers are embedded. Received: 9 August 1996 / Accepted: 23 April 1997     

A model of thrombin inactivation in heparinized and nonheparinized tubes with consequences for thrombus formation

The role of flow and mass transport in determining procoagulant concentration at the wall of synthetic and natural cylindrical blood vessels is analyzed theoretically. The model assumes steady laminar flow and considers, in addition to the fluid dynamic parameters, three rate-determining steps: production of procoagulant (thrombin) and its inactivation at the wall, as well as inactivation in the fluid bulk. The ratio of thrombin wall concentration to production rate Cw/N emerges as a critical parameter in characterizing the behavior of the tube wall. With a wall-inactivation rate typical of heparinized materials, Cw/N = 11.1 s/cm, independent of flow (shear rate) and axial position. This is significantly less than the range of Cw/N (50-500 s/cm) for which the thrombin concentration is high enough to result in significant fibrin formation and thrombosis. Hence little fibrin formation and a high degree of thromboresistance is expected for heparinized materials. Nonheparinized materials have Cw/N values above this range, which are only weakly dependent on shear rate and diameter, suggesting that flow-induced dispersion of thrombin (or other procoagulants) has limited impact on the thrombin wall concentration. These latter results appear to refute the conventional wisdom that attributes the relative patency of large-diameter vessels and differences between venous and arterial thrombi to such flow effects. It is likely that additional factors such as flow pulsatility and wall geometry must be considered to account for these observations.     

Smooth muscle cell adhesion on crosslinked hyaluronan gels

Hyaluronic acid (HA)-based polymers (hylans) are highly biocompatible and can be structurally modified to obtain desired mechanical properties. This study evaluated divinyl sulfone-crosslinked solid and particulate hylans as cellular scaffolds. These two hylan types differ in surface characteristics, mode of preparation, HA content, and extent of crosslinking. Neonatal rat aortic smooth muscle cells were cultured on hylan gels coated with matrix factors including collagen I, ECM gel, laminin, and fibronectin and on uncoated controls for < or =4 weeks. Cell attachment was sparse on uncoated controls but significantly enhanced on coated gels. Cell morphology was influenced by the identity of the matrix factors coated and the surface topography of the hylan gels. Cells attached to coated particulate gels appeared either highly spread (collagen, fibronectin) or irregularly shaped (ECM gel, laminin). Cells on laminin and fibronectin-coated solid gels were rounded and nonproliferative. Cells proliferated most rapidly on ECM gel-coated gels. The uneven surface of particulate gels induced more protein deposition and the subsequent attachment and active proliferation of cells. This study shows that surface texturizing and subsequent surface treatment with matrix factors enhances cell attachment and proliferation of hylans. These results are useful toward developing bioengineered materials based on cell-hylan composites.     

Modulatory effects of rat endothelin on central cardiovascular control in rats

To characterize the modulatory action of rat endothelin (endothelin-3 or ET-3) on the cardiovascular control by the central nervous system (CNS), ET-3 was injected into the cisterna magna of urethane-anesthetized and immobilized rats. An injection of 100 pmol of ET-3 caused immediate rises in arterial pressure (AP), renal nerve activity (RNA), and heart rate (HR). These variables subsequently decreased and, in 5-20 min, fell below the pre-injection level. Simultaneously, the arterial baroreceptor reflex was almost totally suppressed. Although RNA and HR subsequently returned to, or often exceeded, pre-injection levels in 20 to 60 min and reflex activity recovered, AP sometimes remained below control for at least 2 h. A similar pattern of changes was elicited in unanesthetized precollicular decerebrated rats. The responses to ET-3 were abolished by hexamethonium chloride, but were not conspicuously altered by arginine vasopressin antagonist or angiotensin II antagonist. The CNS sites responsible for ET-induced changes were subsequently searched. Topical application of ET-3 to the ventral surface of the medulla (VSM) caused the pattern of changes in AP, RNA, and HR similar to that following intracisternal injection. Microinjection of ET-3 into the nucl. tractus solitarius (NTS) increased AP and RNA, whereas intrathecal administration of it decreased them. We conclude that intracisternally administered ET-3 centrally modulates both tonic and reflex control of AP by the sympathetic nervous system and that the VSM appears to be primarily responsible for the modulation, although NTS and spinal cord may also be involved in it.     

Hylan G-F 20 induces delayed foreign body inflammation in Guinea pigs and rabbits

Recent clinical evidence suggests that hylan, a modified hyaluronan, and related products potentially elicit foreign body granulomatous inflammation in human soft tissue. We investigated the biocompatibility of hylan G-F 20 (Synvisc) for up to 28 days after intradermal injection in guinea pigs and intramuscular injection in rabbits. Compared to saline and unmodified hyaluronan, hylan induced definitive macroscopic changes in guinea pigs by day 14 or later and in rabbits by 28 days after injection. Histologically, at the injection sites, there was severe granulomatous inflammation in guinea pigs and acute inflammation with minimal infiltration of macrophages and foreign body giant cells in rabbits. Furthermore, specific antibodies against hylan were demonstrated in guinea pigs by passive cutaneous anaphylaxis, and substantial deposits of IgG on hylan were evident by immunohistochemistry. The present results contradict previous reports on biocompatibility of hylan and suggest that hylan may potentially induce similar unfavorable reactions in humans.     

Protective effect of fibrin on tumour metastasis

As a potential explanation for the association between metastatic characteristics of tumour cells and their ability to induce fibrin formation, we hypothesize that fibrin deposition around tumour cells might prevent their recognition and lysis by natural killer (NK) or other cytotoxic effector cells. As the hypothesis predicts, we find that both anticoagulants (heparin or warfarin) and immune system stimulants (poly LC) inhibit formation of metastatic nodules in lungs of mice injected with tumour cells through the tail vein. Anticoagulants have no protective effect, however, in mice whose NK cells have been depleted by pretreatment with anti-asialo GM1 serum.Measurement of the elimination of freshly injected radiolabelled tumour cells from the lungs show that this rate is increased by both anticoagulants and immune system stimulation. Again, however, anticoagulants have no effect in NK-depleted animals. That this is due to fibrin deposition, not other blood elements, is confirmed by in vitro experiments: The ability of NK cells to kill tumour cells is inhibited when either cell type is premixed with fibrinogen and thrombin 20 min before the other cell type is added.     

Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels

Fibrin is a biopolymer that has been used in a variety of biomaterial, cell delivery and tissue engineering applications. The enzyme thrombin catalyzes the formation of fibrin microfibrils, which form a three-dimensional mesh in which cells can be directly embedded at the time of gel formation. In this study, fibrin hydrogels containing vascular smooth muscle cells were created using varying concentrations of thrombin. Over 7 days in culture, all gels decreased in volume as the fibrin matrix compacted, and the degree of gel compaction increased as thrombin concentration decreased. The material modulus and ultimate tensile stress of the gels also increased with decreasing thrombin concentration. Addition of thrombin to similar constructs made using collagen Type I did not show an effect on gel compaction or mechanical properties, suggesting that these effects were a result of thrombin's action on fibrin polymerization, and not cellular functions. Cell proliferation in fibrin hydrogels was not significantly affected by thrombin addition. Matrix examination using scanning electron microscopy showed increasing fibrin fiber diameters as thrombin concentration decreased. Confocal microscopic imaging of the actin cytoskeleton showed that cell morphology on two-dimensional substrates of fibrin showed marked changes, with higher thrombin concentrations producing cells with longer cellular projections. However, these morphological changes were not as apparent in cells embedded in three-dimensional (3-D) matrices, in which cells exhibited a similar morphology independent of thrombin concentration. These results relate features of the matrix and cellular components of 3-D fibrin constructs to mechanical properties, and contribute to the understanding of structure-function relationships in cell-seeded, 3-D protein hydrogels.     

Neuronal nitric oxide synthase and splanchnic blood flow in anaesthetized rats

AIMS: To evaluate to what extent the neuronal form of constitutive nitric oxide synthase (nNOS) contributes to the blood perfusion of splanchnic organs, including the islets of Langerhans. METHODS: The nNOS inhibitor 7-nitroindazole (300 mg kg(-1) i.p.) was administered to anaesthetized Sprague-Dawley rats, some of which were pre-treated with the ganglionic blocker hexamethonium (20 mg kg(-1) i.v.) The blood perfusion of the splanchnic organs, including the pancreatic islets was then measured with a microsphere technique. RESULTS: Nitroindazole decreased total pancreatic, duodenal and renal blood flow, whereas pancreatic islet, colonic and adrenal blood flows were unchanged. A slight increase in mean arterial blood pressure was seen after nitroindazole treatment. Nitroindazole did not affect blood glucose or serum insulin concentrations. In separate experiments, hexamethonium affected none of the studied blood flow values, suggesting that the effects of nNOS-inhibition were not mediated from the nervous system. CONCLUSION: Nitric oxide derived from the activity of nNOS contributes to the blood perfusion in the upper portions of the gastrointestinal tract, viz. the parts supplied by the cranial mesenteric artery, and the kidneys, whilst no effects are seen on colonic or adrenal blood flow. Pancreatic islet blood flow was unaffected by nNOS inhibition, thereby suggesting that NO derived from the other isoforms of NOS maintains the high basal islet blood perfusion.     

Injectable mesenchymal stem cell therapy for large cartilage defects--a porcine model

Current techniques in biological resurfacing of cartilage defects require an open arthrotomy or arthroscopy and involve the direct transplantation of isolated cells and/or scaffolds or whole tissue grafts with chondrogenic potential onto the cartilage defect. Our study investigates the possibility of direct intra-articular injection of mesenchymal stem cells suspended in hyaluronic acid (HA) as an alternative to the much more invasive methods currently available. A partial-thickness (without penetration of the subchondral bone) cartilage defect was created in the medial femoral condyle of an adult minipig. Mesenchymal stem cells from the iliac crest marrow of the same pig harvested in a separate procedure and suspended in 2 milliliters of hylan G-F 20 (Synvisc) were injected intra-articularly after the creation of the defect. This was followed by two more injections of hylan G-F 20 (HA) at weekly intervals. Either saline or HA was injected into the knees of the controls. The pigs were sacrificed at 6 and 12 weeks for morphological and histological analysis. The cell-treated groups showed improved cartilage healing both histologically and morphologically at 6 and 12 weeks compared with both controls. The use of intra-articular injections of mesenchymal stem cells suspended in HA is a viable option for treating large cartilage defects. This would be further explored in clinical trials.     

Ring formation of the internal iliac artery

We observed an arterial ring communicating the superior and inferior gluteal arteries in the left half of the pelvis of an 88-year-old male. Although many previous studies have shown variations in the internal iliac artery, there has been no literature describing the fenestration. Therapeutic embolization is commonly performed for intractable bleeding in pelvic region. Surgeons should be aware of the arterial ring formation because of possible danger in the intravascular treatments. In patients with similar arterial rings, embolization of the anterior trunk of the internal iliac artery could be insufficient when blood runs through the circle of the arterial ring.     

Lubrication and wear properties of grafted polyelectrolytes, hyaluronan and hylan, measured in the surface forces apparatus

Hyaluronan is believed to have an important function in the boundary biolubrication of articular cartilage. Using a Surface Forces Apparatus, we tested the tribological properties of surface bound, rather than "free" hyaluronan. The grafting process of the polyelectrolyte included either a biological route via an HA-binding protein or a chemical reaction to covalently bind the polymer to a lipid bilayer coated surface. In another reaction, we constructed a surface with covalently grafted hylan (crosslinked hyaluronan). We studied the normal and shear forces between these surfaces. None of the systems demonstrated comparable lubrication to that found between cartilage surfaces except at very low loads. Both grafted hyaluronan and hylan generated coefficients of friction between 0.15 and 0.3. Thus, the polysaccharide, which is a constituent of the lamina splendens (outermost cartilage layer), is not expected to be the responsible molecule for the great lubricity of cartilage; however, it may contribute to the load bearing and wear protection of these surfaces. This was concluded from the results with hylan, where a thin gel layer was sufficient to shield the underlying surfaces from damage even at applied pressures of over 200 atmospheres during shear. Our study shows that a low coefficient of friction is not a requirement for, or necessarily a measure of, wear protection.     

A mathematical model of thrombin production in blood coagulation,Part I: The sparsely covered membrane case

This paper presents the first attempt to model the blood coagulation reactions in flowing blood. The model focuses on the common pathway and includes activation of factor X and prothrombin, including feedback activation of cofactors VIII and V by thrombin, and plasma inhibition of factor Xa and thrombin. In this paper, the first of two, the sparsely covered membrane (SCM) case is presented. This considers the limiting situation where platelet membrane binding sites are in excess, such that no membrane saturation or binding competition occurs. Under these conditions, the model predicts that the two positive feedback loops lead to multiple steady-state behavior in the range of intermediate mass transfer rates. It will be shown that this results in three parameter regions exhibiting very different thrombin production patterns. The model predicts the effect of flow on steady-state and dynamic thrombin production and attempts to explain the difference between venous and arterial thrombi. The reliance of thrombin production on precursor procoagulant protein concentrations is also assessed.