Liver Support Systems

Morphology of the Liver

Liver Functions

Hepatic Failure

Pierre M. Galletti

(deceased)

Hugo O. Jauregui

Rhode Island Hospital

подпись: pierre m. galletti
(deceased)
hugo o. jauregui
rhode island hospital
Liver Support Systems

Global Replacement of Liver Function

Hybrid Replacement Procedures

Source of Functional Cells • Supporting Structures

Outlook

Morphology of the Liver

The liver is a complex organ that operates both in series and in parallel with the gastrointestinal tract. After entering the portal system, the products of digestion come in contact with the liver parenchymal cells, or hepatocytes, which remove most of the carbohydrates, amino acids, and fats from the feeder circulation, therefore preventing excessive increases throughout the body after a meal. In the liver, these products are then stored, modified, and slowly released to the better advantage of the whole organism.

The liver can be considered a complex large-scale biochemical reactor, since it occupies a central position in the metabolism, i. e., the sum of the physical and chemical processes by which living matter is produced, maintained, and destroyed, and whereby energy is made available for the functioning of liver cells as well as tissues from all other organs.

The adult human liver (weighing 1500 g) receives its extensive blood supply (on the order of 1 L/min or 20% of cardiac output) from two sources: the portal vein (over two-thirds) and the hepatic artery (about one-third). Blood from the liver drains through the hepatic veins into the inferior vena cava. Macroscopically, the liver is divided into 4 or 5 lobes with individual blood supply and bile drainage channels. Some of these lobes can be surgically separated, although not without difficulty.

Microscopically, human hepatocytes (250-500 □ 109 in each liver) are arranged in plates (Fig. 133.1) that are radially distributed around the central (drainage) vein [Jones & Spring-Mills, 1977] and form somewhat hexagonal structures, or liver lobules, which are much more clearly demarcated in porcine livers. Present in the periphery of these lobules are the so-called portal triads, in the ratio of three triads for each central vein. In each portal triad, there are tributaries of the portal vein, branches of the hepatic artery, and collector ducts for the bile (Fig. 133.1). BLood enters the liver lobule at the periphery from terminal branches of the portal vein and the hepatic arteries and is distributed into capillaries which separate the hepatocyte plates. These capillaries, called sinusoids, characteristically have walls lined by layers of endothelial cells that are not continuous but are perforated by small holes (fenestrae). Other cells are present in the sinusoid wall, e. g., phagocytic Kuppfer cells, fat-storing Ito cells, and probably a few yet undefined mesenchymal cells. It is important to emphasize that blood-borne products (with the exception of blood cells) have free access to the perisinusoidal space, called the space of Disse, which can be visualized by electron microscopy as a gap separating the sinusoidal wall from the hepatocyte plasma

Liver Support Systems

FIGURE 133.1 The liver lobule.

FENESTHAE HEPATOCYTES

Liver Support Systems

FENESTRAE Bill DdnSllCUlt

FIGURE 133.2 Hepatocyte relationships with the space of Disse and the sinusoid wall.

Membrane (Fig. 133.2). In this space, modern immunomicroscopic studies have identified three types of collagens: Type IV (the most abundant), Type I, and Type III. Fibronectin and glycosaminoglycans are also found there, but laminin is only present in the early stages of liver development not in adult mammalian livers [Martinez-Hernandez, 1984].

The hepatocytes themselves are large (each side about 25 microns), multifaceted, polarized cells with an apical surface which constitutes the wall of the bile canaliculus (the channel for bile excretion) and basolateral surfaces which lie in close proximity to the blood supply. Hepatocytes constitute 80-90% of the liver cell mass. Kuppfer cells (about 2%) belong to the reticulo-endothelial system, a widespread class of cells which specialize in the removal of particulate bodies, old blood cells, and infectious agents from the blood stream.

The cytoplasm of hepatocytes contains an abundance of smooth and rough endoplasmic reticulum, ribosomes, lysosomes, and mitochondria. These organelles are involved in complex biochemical pro­cesses: fat and lipid metabolism, synthesis of lipoproteins and cholesterol, protein metabolism, and synthesis of complex proteins, e. g., serum albumin, transferrin, and clotting factors from amino acid building blocks. The major aspects of detoxification take place in the cisternae of the smooth endoplasmic reticulum, which are the site of complex oxidoreductase enzymes known collectively as the cytochrome P-450 system. In terms of excretion, hepatocytes produce bile, which contains bile salts and conjugated products. Hepatocytes also store large pools of essential nutrients such as folic acid, retinol, and cobalamin.

Liver Functions

The liver fulfills multiple and finely tuned functions that are critical for the homeostasis of the human body. Although individual pathways for synthesis and breakdown of carbohydrates, lipids, amino acids, proteins, and nucleic acids can be identified in other mammalian cells, only the liver performs all these biochemical transformations simultaneously and is able to combine them to accomplish its vital biologic task. The liver is also the principal site of biotransformation, activation or inactivation of drugs and synthetic chemicals. Therefore, this organ displays a unique biologic complexity. When it fails, functional replacement presents one of he most difficult challenges in substitutive medicine.

Under normal physiologic requirements, the liver modifies the composition and concentration of the incoming nutrients for its own usage and for the benefit of other tissues. Among the major liver functions, the detoxification of foreign toxic substances (xenobiotics), the regulation of essential nutrients, and the secretion of transport proteins and critical plasma components of the blood coagulation system are probably the main elements to evaluate in a successful organ replacement [Jauregui, 1991]. The liver also synthesizes several other critical proteins, excretes bile, and stores excess products for later usage, functions that can temporarily be dispensed with but must eventually be provided.

The principal functions of the liver are listed in Table 133.1. The challenge of liver support in case of organ failure is apparent from the complexity of functions served by liver cells and from our still imperfect ability to rank these functions in terms of urgency of replacement.

TABLE 133.1 Liver Functions

Carbohydrate metabolism: Glyconeogenesis and glycogenolysis Fat and lipid metabolism: Synthesis of lipoproteins and cholesterol Synthesis of plasma proteins, for example:

Albumin Globulins Fibrinogen Coagulation factors Transferrin □ — fetoprotein

Conjugation of bile acids; conversion of heme to bilirubin and biliverdin

Detoxification: Transformation of metabolites, toxins, and hormones into water-soluble compounds (e. g., cytochrome P-450 P-450 oxidation, glucuronyl transferase conjugation)

Biotransformation and detoxification of drugs Metabolism and storage of vitamins Storage of essential nutrients Regeneration

Hepatic Failure

More than any other organ, the liver has the property of regeneration after tissue damage. Removal or destruction of a large mass of hepatic parenchyma stimulates controlled growth to replace the missing tissue. This can be induced experimentally, e. g., two thirds of a rat liver can be excised with no ill effects and will be replaced within 6 to 8 days. The same phenomenon can be observed in humans and is a factor in the attempted healing process characteristic of the condition called liver cirrhosis. Recent attempts at liver transplantation using a liver lobe from a living donor rely on the same expectation of recovery of lost liver mass. Liver regeneration is illustrated by the myth of Prometheus, a giant who survived in spite of continuous partial hepatectomy through the good auspices of a vulture (a surgical procedure inflicted on him as punishment for having stolen the secret fire from the gods and passing it on to humanity).

Hepatic failure may be acute or chronic according to the time span it takes for the condition to develop. Mechanisms and toxic by-products perpetuating these two conditions are not necessarily the same. Acute fulminant hepatic failure (FHF) is the result of massive necrosis of hepatocytes induced over a period of days or weeks by toxic substances or viral infection. It is characterized by jaundice and mental confusion which progresses rapidly to stupor or coma. The latter condition, hepatic encephalopathy (HE), is currently thought to be associated with diminished hepatic catabolism. Metabolites have been identified which impair synaptic contacts and inhibit neuromuscular and mental functions (Table 133.2). Although brain impairment is the rule in this condition, there is no anatomic damage to any of the brain structures, and therefore, the whole process is potentially reversible. The mortality rate of FHF is high (70-90%), and death is quite rapid (a week or two). Liver transplantation is currently the only effective form of treatment for FHF. Transplantation procedures carried out in life-threatening circumstances are much more risky than interventions in relatively better compensated patients. The earlier the transplantation procedure takes place, the greater is the chance for patient survival. However, 10-30% of FHF patients will regenerate their liver under proper medical management without any surgical intervention. Hence, liver transplan­tation presents the dilemma of choosing between an early intervention, which might be unnecessary in some cases, or proceeding to a late procedure with a statistically higher mortality [Jauregui et al., 1994].

Chronic hepatic failure, the more common and progressive form of the disease, is often associated with morphologic liver changes known as cirrhosis in which fibrotic tissue gradually replaces liver tissue as the result of long-standing toxic exposure (e. g., alcoholism) or secondary to viral hepatitis. More than

People died of liver failure in the United States in 1990.

In chronic hepatic failure, damaged hepatocytes are unable to detoxify toxic nitrogenous products that are absorbed by intestinal capillaries and carried to the liver by the portal system. Ammonia probably plays the major role in the deterioration of the patient’s mental status, leading eventually to “hepatic coma.” An imbalance of conventional amino acids (some abnormally high, some low) may also be involved in the pathogenesis of the central nervous system manifestation of hepatic failure, the most dramatic of which is cerebral edema. Impaired blood coagulation (due to decreased serum albumin and clotting factors), hemorrhage in the gastro-intestinal system (increased resistance to blood flow through the liver leads to portal hypertension, ascites formation, and bleeding from esophageal varices), and

TABLE 133.2 Metabolic Products with Potential Effects in Acute Liver Failure

Substance

Mode of action

Ammonia

Neurotoxic interaction with other neurotransmitters Contributes to brain edema

Benziodiazepinelike substances

Neural inhibition

GABA

Neural inhibition

Mercaptans

Inhibition of Na-K ATPase

Octopamine

Acts as a false neurotransmitter

Hepatic encephalopathy with glial cell damage in the brain are the standard landmarks of chronic hepatic failure. In fact, HE in chronic liver failure is often precipitated by episodes of bleeding and infection, and progression to deep coma is an ominous sign of impending death.

Intensive management of chronic liver failure includes fluid and hemodynamic support, correction of electrolyte and acid-base abnormalities, respiratory assistance, and treatment of cerebral edema if present. Aggressive therapy can diminish the depth of the coma and improve the clinical signs, but the outcome remains grim. Eventually, 60-90% of the patients require transplantation. About 2500 liver transplants are performed every year in the United States, with a survival rate ranging from 68-92%. The most serious limitation to liver transplantation (besides associated interrelated diseases) remains donor scarcity. Even if segmented transplants and transplants from living related donors become acceptable practices, it is unlikely that the supply of organs will ever meet the demand. Further, the problem of keeping a patient alive with terminal hepatic failure, either chronic or acute, while waiting for an adequately matched transplant is much more difficult than the parallel problem in end-stage renal disease, where dialysis is a standardized and effective support modality.

An appreciation of the modalities of presentation of the two types of hepatic coma encountered in liver failure is needed for a definition of the requirements for the proper use of liver assist devices. In the case of FHF, the hepatologist wants an extracorporeal device that will circulate a large volume of blood through a detoxifying system [Jauregui & Muller, 1992] allowing either the regeneration of the patient’s damaged liver (and the avoidance of a costly and risky liver transplantation procedure) or the metabolic support needed for keeping the patient alive while identifying a cadaveric donor organ. In the first option, the extracorporeal liver assist device functions as an organ substitute for the time it takes the liver to regenerate and recover its function; in the second, it serves as temporary bridge to transplantation.

In the case of chronic liver failure today, spontaneous recovery appears impossible. The damaged liver needs to be replaced by a donor organ, although not with the urgency of FHF. The extracorporeal liver assist device (LAD) is used as a bridge while waiting for the availability of a transplant. It follows that the two different types of liver failure may require different bioengineering designs.

Liver Support Systems

The concept of artificial liver support is predicated on the therapeutic benefit of removing toxic substances accumulating in the circulation of liver failure patients. These metabolites reflect the lack of detoxification by damaged hepatocytes, the lack of clearance of bacterial products from the gut by impaired Kupffer cells, and possibly the release of necrotic products from damaged cells which inhibit liver regeneration. Systemic endotoxemia as well as massive liver injury give rise to an inflammatory reaction with activation of monocytes and macrophages and release of cytokines which may be causally involved in the patho­genesis of multiorgan failure commonly encountered in liver failure.

Technologies for temporary liver support focus on the detoxifying function, since this appears to be the most urgent problem in liver failure. The procedures and devices which have been considered for this purpose include the following.

Hemodialysis

Hemodialysis with conventional cellulosic membranes (cut-off point around 2000 daltons) or more permeable polysulfone or polyacrylonitrile [de Groot et al., 1984] (cut-off 1500-5000 daltons) helps to restore electrolyte and acid-base balance and may decrease the blood ammonia levels but cannot remove large molecules and plasma protein-bound toxins. Improvement of the patient’s clinical condition (e. g., amelioration of consciousness and cerebral edema) is temporary. The treatment appears to have no lasting value and no demonstrated effect on patient survival. In addition, hemodialysis may produce a respiratory distress syndrome caused by a complement-mediated poly-morphonuclear cell aggregation in the pulmonary circulatory bed. Because some of the clinical benefit seems related to the removal of toxic molecules, more aggressive approaches focused on detoxification have been attempted.

Hemofiltration with high cut-off point membranes (around 50,000 daltons with some polyacrylonitrile- polyvinyl chloride copolymers, modified celluloses, or polysulfones) clears natural or abnormal com­pounds within limits imposed by convective transport across the exchange membrane. These procedures again have a temporary favorable effect on hepatic encephalopathy (perhaps because of the correction of toxic levels of certain amino acids) with reversal of coma, but they do not clearly improve survival rates.

[1]An alternate form of this equation, which accounts more rigorously for transfer of drug between different phases in the tissue, is also available [19].,

The fundamental aspects cf cell engineering are twofold: (1) quantitative understanding cf cell function in molecular terms and (2) ability tc manipulate cell function through molecular mechanisms, whether

Extracellular matrix proteins such as fibronectin, laminin, vitronectin and collagen, or adhesion mole­cules such as ICAM-1, VCAM-1, PCAM-1, and sialyl Lewis X, interact with cell surface receptors and mediate cell adhesion. The tripeptide adhesion sequence Arg-Gly-Asp (RGD) is a ubiquitous signal present in many cell adhesion proteins. It interacts with the integrin family of cell surface adhesion receptors, and comprises the best studied ligand-receptor pair [94-96]. In lieu of immobilizing complex multifunctional proteins for purposes of cell adhesion studies, synthetic RGD sequences have instead been immobilized onto many substrates as simplified models to understand various molecular aspects of cell adhesion phenomena. The following paragraphs cite examples of RGD-grafted substrates that have been used in biomedicine and bioengineering.

The density of RGD necessary to mediate cell adhesion has been determined in a number of fashions. RGD-containing peptides and protein fragments have been physicochemically adsorbed onto tissue culture substrates [97, 98] or covalently bound to albumin-coated substrates [45, 46] to titrate the dependency of cell adhesion function upon RGD surface densities. To remove potential complications due to desorption of ligands or albumin, RGD has been covalently bound onto functionalized substrates. Immobilization also restricts the number of conformations the peptide may assume, helping to ensure that all the peptide is accessible to the cells. RGD has been immobilized onto silanated glasses by its amino [19, 99] and carboxyl [15] termini. The effects of RGD density on cell adhesion, spreading, and cytoskeletal organization was examined [99] using this well-defined system. Other peptides have been immobilized in identical fashion to determine if they influence cell physiology [100].

RGD peptides have been immobilized onto highly cell-resistant materials to ensure that the peptide is the only cell adhesion signal responsible for cell adhesion to diminish signals borne of nonspecifically adsorbed serum proteins. Hydrogels of polyacrylamide [101], poly(vinyl alcohol) [31] and poly(ethylene glycol) [85] and nonhydrogel networks of polyacrylate/poly(ethylene glycol) [102] have been grafted with RGD; these background materials were highly resistant to the adhesion of cells even in the presence of serum proteins, demonstrating that the RGD sequence was solely responsible for mediating cell adhesion.

RGD-containing peptides have been immobilized onto medically relevant polymers in an effort to enhance their biocompatibilities by containing an adhered layer of viable cells. RGD-grafted surfaces can be more efficient in supporting the number and strength of cell adhesion by the peptide facilitating cell adhesion additionally to adsorbed adhesion proteins from the biological milieu. RGD has been conjugated

[4]For more complete definitions see the Oxford Dictionary of Biochemistry and Molecular Biology, 1997.

Spleen. Apparently, the splenic microenvironment can at least transiently support stem cell growth and development.

[6]Injected bone-marrow-derived stem cells lodge and develop in several types of hematopoietic tissue, including

[7] The permeability of the materials which separate the gas phase from the blood phase in the pulmonary alveoli

[8] □ exi

K □

QB Z □ expRQ □ Zl

In a dialyzer, separation occurs because small molecules diffuse more rapidly than larger ones and because the degree to which membranes restrict solute transport usually increases with permeant size (permse — lectivity). Fick’s equation states that solute will move from a region of greater concentration to a region of lower concentration in a rate proportional to the difference in concentration on opposite sides of the membrane

□□D— (130.26)

DX

Where □ = unit solute flux in g/cm2-s; D = diffusion coefficient cm2/s; c = concentration in g/cm3; and x = distance in cm. The minus sign accounts for the convention that flux is considered positive in the direction of decreasing concentration. Diffusion coefficient decreases roughly in proportion to the square root of molecular weight.

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