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Thèse de doctorat/ PhD Thesis Citation APA:

Coessens, B. (s.d.). The role of endothelin production and sensitivity in the no-reflow phenomenon of vascularized bone grafts (Unpublished doctoral dissertation). Université libre de Bruxelles, Faculté de Médecine – Médecine, Bruxelles.

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The Rôle of Endothelin Production and

Sensitivity in the No-Reflow Phenomenon of

Vascularized Bone Grafts

Bruno C. Coessens

UNIVERSITE LlSHiE Î5E BRUXELLES. ■SLIOTHEÜUE DE NILOEGINE ERASME Hüute de Lennik, 808 (Bat. E - CP 607)

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The Rôle of Endoîhelin Production and

Sensitivity in the No-Reflow Phenomenon of

Vascularized Bone Grafts

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à mon père, à ma mère.

au professeur Lejour, qui fut et est toujours l’exemple d’une réussite professionnelle faite d’accomplissement clinique et de rigueur scientifique; la première, elle a éveillé en moi le goût de la recherche,

au professeur De Mey, un chef et un ami, qui m’a soutenu dans cette tâche,

au professeur Wood, un esprit clair et didactique, mais aussi un gentleman qui m’a prodigué conseils et encouragements, et dont la qualité scientifique n’a d’égale que la chaleur de son accueil, au professeur Wei, qui m’a ouvert de nouveaux horizons en microchirurgie,

au professeur Bergmann pour ses avis judicieux dans la rédaction du manuscript,

au docteur Virginia Miller, pour son aide précieuse dans la réalisation de cette recherche,

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to my Father, to my Mother,

to professer Lejour, who was and is still the example of professional success made of clinic accomplishment and scientific rigour; the first, she initiated in me the interest for research,

to professer De Mey, a chief and a friend, who supported me in this task,

to professer Wood, a clear and didactic mind, a gentleman whose advices and encouragements and whose scientific quality are equalled only by the warmth of his welcome,

to professer Wei, who opened before me new perspectives in microsurgery,

to professer Bergmann, for his judicious advices in the writing of the manuscript,

to doctor Virginia Miller, for her precious help in the carrying out of this research,

to Mary Adams, whose support and compétence I did appreciate,

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Résumé

La reconstruction du squelette représente une étape déterminante du

traitement de traumatismes sévères ou de résections tumorales avec

perte de substance osseuse. Les techniques conventionnelles de

greffes d'os sont limitées par la taille de la perte de substance ainsi que

par la qualité vasculaire du site receveur. En effet, le greffon doit être

revascularisé à partir des tissus avoisinants. Les techniques

microchirurgicales permettent actuellement la reconstruction en un

temps opératoire des solutions de continuité osseuses étendues et cela

même en terrain mal vascularisé par la restauration de la

vascularisation propre de l'os.

Différentes études cliniques ont établi que les greffes osseuses

vascularisées s'incorporent nettement mieux que les greffes

conventionnelles dans le type de situation décrit plus haut.

Cependant, le nombre d'ostéocytes viables ainsi que le flux sanguin

interosseux y sont moindres que dans l'os normal. Des altérations

dans l'apport sanguin peuvent ainsi être responsables d'un taux

d'échecs allant jusqu'à 15% et d'une incidence élevée de retard de

consolidation.

Ces observations pourraient être imputables à une augmentation des

résistances vasculaires périphériques avec, pour conséquence, une

perfusion inadéquate du transplant malgré des anastomoses

perméables. Ce phénomène, appellé "no-reflow", est défini comme

une obstruction périphérique au flux sanguin et semble lié à la période

d'ischémie.

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sanguin osseux de l'endotheline, un autre produit de l'endothelium

vasculaire, n’est quand à lui que très partiellement documenté.

L'endotheline (ET) représente une famille d'hormones paracrines

munies de puissantes propriétés vasoactives. Elles sont présentes sous

au moins trois isoformes (ET-1, ET-2 et ET-3); ET-1 seulement serait

produit au niveau de l’endothelium vasculaire. ET-1 est formée à partir

d'un précurseur de 38 acides aminés appelé "big endothelin", ensuite

clivé en un peptide de 21 acides aminés qui représente la forme active.

Il induit dans certains lits vasculaires une vasodilatation à doses basses

et, lorsque les doses augmentent, produit uniformément une

vasoconstriction prolongée. Il s'agit d’un des plus puissants

vasoconstricteurs produits de façon endogène. L'activité

vasodilatatrice initiale paraît provenir de l'endothélium; sa source

dépendrait de la production de prostacycline et d’oxyde nitrique.

Deux sous-types de récepteurs pour l’endotheline ont pu être identifiés

et clonés, ils sont classés ET-A et ET-B. Le récepteur ET-A, localisé

au niveau du muscle vasculaire hsse présente une très forte affinité

pour ET-1 et seulement une faible affinité pour ET-3. C’est par son

intermédiaire que se produit la majorité de l’action constrictive de

l'endotheline au niveau du muscle vasculaire lisse. Les récepteurs ET-

B, moins spécifiques, partagent une affinité égale pour les différentes

isoformes de l'endotheline. Le récepteur ET-B, qui est localisé au

niveau de l'endothelium vasculaire semble induire l'action

vasodilatatrice de l'endotheline.

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Ce travail à pour but de documenter le rôle de l'endotheline au niveau

des mécanisme de contrôle du flux sanguin dans l'os. Ce type

d'information permettra une meilleure compréhension des altérations

de l'apport vasculaire dans les greffes osseuses vascularisées ainsi que

du rôle de l'endotheline dans la pathologie du "no-reflow". Enfin cette

étude pourra fournir des informations sur les techniques de

préservation de l'os vascularisé dans la perspective d'allotransplants.

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l'endotheline présente uniquement une action vasoconstrictrive au

niveau osseux, un effet médié par les récepteurs ET-A seuls.

Dans la seconde partie, l'influence de l'ischémie froide avec ou sans

microperfusion avec la solution de l'Université du Wisconsin, sur la

production d’ET-1 et sur les récepteurs ET est étudiée. Le modèle de

perfusion in vitro du tibia est utilisé afin d'isoler l'endothelium

vasculaire des éléments figurés du sang. La production endogène

d'ET-1 n'est pas influencée par une ischémie froide de 24 H. La

sarafotoxine S6c ne produit toujours aucune réponse dans ces

conditions. Par contre la réponse induite par les récepteurs ET-A est

significativement augmentée. Cette observation n'est pas influencée

par la technique de préservation. L'altération de la réponse des

récepteurs ET-A représente la seule modification de la fonction

paracrine de l'endothelium observée après 24 H d'ischémie froide dans

le modèle étudié. Compte tenu des propriétés pharmacologiques de

l'endotheline (vasoconstriction puissante et de longue durée), cette

observation pourrait expliquer l'origine du vasospasme in vivo.

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production d'oxide nitrique et de la synthèse de prostacycline

n'influence pas l'effet de l'acidose sur les contractions produites par le

KCL. Cependant, l'inhibiteur sélectif des récepteurs ET-A, BQ123,

qui à Ph normal n'a pas d'influence sur les réponses au KCL, supprime

complètement l’atténuation de l'effet constricteur du KCL observé lors

de l'acidose. Cette observation lie les récepteurs ET-A à la réponse de

l'endothelium vasculaire de l'artère nourricière du tibia dans des

conditions d'acidose.

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TABLE of CONTENTS

n

ous voyons

Part 1. ANATOMY and PHYSIOLOGY of VASCULARIZED

BONE GRAFT

pp: 6-51

1- Introduction:

pp: 6- 7

2- Vascular Anatomy of Long Bones

pp: 8-15

2.1- History pp: 8- 9

2.2- Arterial Suppiy

2.21- Nutrient Artery 2.22- Periosteal Arteries

2.23- Epiphyseal and Metaphyseal Arteries

pp: 9-11 pp: 9-10

p: 10 pp: 10-11 2.3- The Microdrculation of the Bone

2.31- The Martow Microcirculation 2.32- The Cortical Microcirculation

pp: 11-13 pp: 12-13

p: 13

2.4- Venons Drainage pp: 13-14

2.5- Lymphatics p: 15

3- Control Mechanisms of the Bone Blood Flow pp;

15-33

3.1-lntroduction pp: 15-16

3.2- Neuronal Mechanisms pp: 16-17

3.3- Humoral Factors pp: 17-20

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pp: 21-33 3.5- Endothélial Cell Mechanism

3.51- The concept of Endothélial Régulation of

Vascular Tone pp: 21-22

3.52- Vasodilator Prostaglandins pp: 22-25 3.53- Endothelium-Derived Relaxing Factor pp: 25-27

3.54- Endothelin pp; 27-33

3.541- Pressor Actions pp: 28-29 3.542- Endothelin Receptors in the

vascular tissue pp; 29-32

3.543- Rôle of Endothelin pp; 32-33

4- Vascularized Bone Grafts

pp: 34-39

4.1- Development of Vascularized Bone Grafts p: 34 4.2- Healing Mechanism of Non-Vascularized

Bone Grafts pp: 34-35

4.3- Healing and Remodeling of Vascularized

Bone Grafts pp: 35-36

4.4- Clinical Applications in

Mandibular Reconstruction pp: 36-41

4.41- Introduction pp: 36-37

4.42- Flap Sélection p: 38

4.43- Donor Site Review pp: 38-40

4.44- Fixation and Shaping of the Bone pp: 40-41

4.45- Conclusion p: 41

5- Ischémie Injury and Préservation of Vascularized Bone

Grafts

pp: 42-51

5.1- Introduction p: 42

5.2- The No-Reflow Phenomenon and Reperfusion

Injury pp: 42-47

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of Reperfusion Injury pp: 46-47

5.3- Organ Préservation pp: 47-50

5.31- Cold Storage pp: 47-49

5.32- Continuous Hypothermie Perfusion p: 49

5.33- Cryopreservation p: 50

5.4- Préservation of Vascularized Bone Grafts pp: 50-51

Parti EXPERIMENTAL STUDY

pp: 52-101

6- Aim

p: 52

7- Experimental Methods

pp: 53-61

7.1- Organ Chamber Study

7.11- The Model

7.12- Optimal Length Tension

7.13- Détermination of Endothélium Dependence

pp: 53-55

p: 54 p: 54 pp: 54-55

7.2- The In Vitro Organ Perfusion

7.21- The Vascular Anatomy of the Canine Tibia 7.22- Isolation of the Canine Tibia

7.23- The Perfusion Apparatus

7.24- Détermination of Optimal Flow Rate

pp: 55-59 p: 56 p: 57 pp: 57-59 p: 59 7.3- EndotheUn-1^ Assay pp: 60-61

8- Characterization of the Effects of Endothelins and Receptors

in both Isolated Nutrient Vessels of the Canine Tibia and in

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8.1- Introduction p:62

8.2- Aim p; 63

8.3- Material and Metiiods pp: 63-66

8.31- Animais p: 63 8.32- Drugs p: 63 8.33- Organ Chamber p: 64 8.331- Experimental Design p; 64 8.34- Bone Perfusion p: 64 8.341- Experimental Design pp: 64-65 8.35- Data Analysis pp: 65-66 8.4- Results pp: 66-74 8.41- Organ Chamber pp: 66-69 8.42- Bone Perfusion pp: 70-74 8.5- Discussion pp: 75-76

Evaluation of the Influence of 24 H Cold Préservation on

Endothelin Production and on Endothelin Receptors in the

Bone Vasculature

pp: 77-88

9.1- Introduction p; 77

9.2- Aim pp: 77-78

9.3- Material and Methods pp: 78-81

9.31- Animais p: 78

9.32- Drugs p: 78

9.33- Bone Perfusion pp: 78-79

9.331- Cold Storage p: 78

9.332- Cold Storage with Microperfusion

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10-

Evaluation of the Effects of Acidosis on ET-A Receptors in

the Canine Tibia Nutrient Artery

pp: 89-99

10.1- Introduction p: 89

10.2- Aim p; 89

10.3- Material and Methods pp: 90-92

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Part 1. ANATOMY and PHYSIOLOGY of VASCULARIZED

BONE GRAFTS

1- Introduction

Skeletal reconstruction with bone grafting is an increasingly important aspect of the reconstructive surgery in dealing with bone loss secondary to severe trauma or tumor resection. Conventional techniques of bone grafting can be used in the vast majority of cases, but their efficacy for the treatment of large segmentai bone defects particularly in a poorly vascularized environment is often limited. Advances in microvascular surgery hâve made it possible to reconstitute these defects with autogenous bone grafts revascularized by microsurgical techniques. The nutrient blood supply to the bone is restored through anastomoses between its vascular pedicle and artery and veins in the récipient area . With the nutrient blood supply preserved, osteoclasts and ostéocytes within the graft may survive and participate directly in the healing process.

Clinical studies hâve suggested that these grafts hâve superior rates of healing in comparison with non-vascularized bone grafts. However, experimental studies indicate that vascularized grafts contain decreased numbers of viable ostéocytes (Arata, 1984) and despite patent vascular anastomoses, blood flow is lower than in normal bone {Siegert et ai, 1990).

Numerous mechanisms may lead to the failure of reperfusion in vascularized bone grafts, such as the no-reflow phenomenon, ischémie reperfusion injury, and alteration of the normal vascular control mechanisms. In order to study these possible changes in bone, it is necessary to more clearly understand the blood flow regulating mechanisms.

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implicated as having a rôle in conditions associated with tissue ischemia, and may be involved in the failure of tissue reperfusion after a period of ischemia. However, the effects of endothelin on the bone vasculature is only poorly documented.

Little is known about the type of receptors présent in the bone vasculature as well as the type of response to ET-1 in both the fresh and preserved bone vasculature. The aim of this study is to détermine the effects of endothelin in the isolated nutrient vessel of the canine tibia as well as in perfused canine tibial bones in regards to its possible rôle in the vascular control mechanisms of revascularized bone grafts. The interaction of ET-1 with other known pathophysiological mechanisms is also studied.

For the surgeon the relevance of research focusing on ischémie damage extends beyond today's free tissue transfer to the tomorrow's "spare-part surgery". With a more sophisticated understanding of transplantation immunology, the time looms where transfers of stored allogenic bone will be part of our therapeutical armementarium (Friedlaender, 1991). Along with adéquate modulation of immune rejection, it will be necessary to effectively préservé tissues so that they will tolerate prolonged periods of ischemia.

The goal of tissue préservation is to maintain the viability of an organ ex- vivo. Bone is unique to other transplanted organs because survival of the parenchymal cells may not be essential to the success of the graft. If the vascular bed can be maintained, circulating osteoprogenitor cells may "seed" the graft and induce osteogenesis.

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2- Vascular Anatomy of Long Bones

"There is danger inhérent in the mechanical efficiency of our modem methods, danger lets the craftsmen forget that union cannot be imposed but may hâve to be encouraged. For a bone is a plant, with its roots in the soft tissues, and when its vascular connections are damaged, it often requires, not the technique of a cabinet maker, but the patient care and understanding ofa gardener. "

Girdlestone 1932

2.1- History

The first study of the raicrostructure of the bone was reported in 1674 by Antoni Van Leeuwenhoek {Vcm Leeuwenhoek, 1674), the inventor of the microscope. He observed "divers small pipes going longwayes" within the cortex of bones. The same structure was described by Havers in 1691 {Havers, 1691 ) as a System of "straight pores" and were soon referred to as Haversian canals. Havers did not recognize the presence of blood vessels within the canals. This was demonstrated by Albinus in 1754 {Albinus, 1754). The nutrient artery System was well described by Bichat in 1812 (Bichat, 1812), where he raentioned three different "cavités de nutrition". The first description appears to be similar to the nutrient artery, establishing an extemal connection to the bone marrow. The second vascular type was located in the ends of the long bones. It appears that he described the raetaphyseal vessels. The third System to provide nutrition to the cortical bone itself was like the Haversian canals.

The centrifugal arrangement of the Haversian System was first described by Meckel in 1875 {Meckel et ai, 1875).

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Cruveilhier in 1844 (Cruveilhier, 1844) gave an excellent description of the blood supply to the bone and recognized three separate aiterial supplies. This observation was later confirmed by Langer (1876) and Lexer et al. (1904) and is generally accepted today.

2.2- Arterial Supply

Langer and Lexer provided the main outline of the anatomy of the blood supply of long bones. In raammals, regardless of species, long bones hâve three sources of blood supply: one or more nutrient arteries, the periosteal arteries, and the epiphyseal - metaphyseal arteries.

The relative importance of each of these vascular Systems is still controversial. The theory of cortical blood supply presented by Trueta et al.

(Trueta et al., 1955), that the inner two thirds of the cortex is nourished from the

marrow and the outer one third of the cortex from the periosteal vessels is challenged by the concept of the centrifugal blood flow within the cortex

(Brookes, 1971). In a growing animal the territories of supply of the epiphyseal-

metaphyseal arteries are separated from that of the nutrient artery by the epiphyseal plate. At the time of epiphyseal closure the two Systems développe anastomoses. Under normal circumstances it appears that the nutrient and epiphyseal-metaphyseal arteries predominate in supplying the cortex, but under condition of disease or injury any one of these route of supply can increase capacity to provide for the whole bone (Rhinelander, 1968). In a resting State it has been estimated that 70 - 90% of the diaphysis is supplied by the nutrient artery and that it supplies approximately 33% of the metaphyseal and epiphyseal régions

(Shim et al., 1967; MacNab et ai, 1974).

2.21- Nutrient Artery

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several authors {Laing, 1953, 1956; Schulman, 1959; Nelson et ai, 1960; Trias et

ai, 1979; Hallock étal., 1993).

Before entering the bone, the nutrient artery may send several small branches to the periosteum and the adjacent muscles; it does not branch within the nutrient canal. Once in the medullary cavity, the nutrient artery divides into ascending and descending branches. From these, smaller branches stream radially toward and into the endosteal surface of the cortex. Those running centrifugally feed the capillaries of the cortex and Haversian canals (Trias et al., 1979). The others provide the main source of blood for the large System of vessels in the bone marrow (Kelly et ai, 1963; Trueta, 1963; Lopez-Curto étal., 1980)

Within the diaphysis the nutrient artery supply is estimated to be 70% to cortical bone and the remainder to the marrow in adult dogs (Kelly, 1973)

2.22- Periosteal Arteries

Small arteries on the surface of the bone, which originate from the surrounding soft tissue, supply the periosteum of long bones. In areas of soft tissue attachment these arteries may penetrate the cortex. At a capillary level these vessels anastomose with those of the medullary System. Where the overlying soft tissue is of poor vascularity (eg. over the anteromedial cortex of the human tibia) there are few periosteal arteries and these do not penetrate into the cortex (Nelson

et al, 1960; Brookes, 1971). Most workers hâve stressed the importance of the

periosteal vessels in the vascular supply of the cortex (de Mameffe, 1951); however more recent studies fail to confirm their prépondérant rôle under normal conditions (Nelson et al, 1960; Brookes, 1971; Kelly et al, 1990). Periosteal blood supply becomes important with impairment of medullary blood supply. In such instances, large anastomoses can develop between nutrient, periosteal and metaphyseal arteries (Oni étal, 1990b).

2. 23- Epiphyseal and Metaphyseal Arteries

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The epiphyseal arteries penetrate the cortex between the joint capsule and the growth plate supplying the columns of proliferating cartilage cells (.Morgan,

1959). The circumferential supply anses from a small artery that runs around the

periphery of the growth plate. In certain situations (eg. the proximal fémur) the epiphyseal arterial blood supply may be derived entirely from this source {Chung,

1976).

The metaphyseal arteries arise from a plexus of small arteries called the "circulosus articuli vasculosus" (.Hunier, 1743) which surrounds the periosteal surface of the metaphysis. This plexus is fed by articular branches of the major limb vessels and supplies numerous small metaphyseal arteries. These enter the bone in some of the small foramina that characterize the metaphyseal cortex. In the growing bone these vessels only supply the peripheral zone of the metaphysis

(.Trueta et ai, 1960). Subsequently, the metaphyseal arteries become increasingly

prominent. When the growth plate has fused they ramify to supply the whole metaphysis and epiphysis, producing extensive anastomoses between the nutrient and metaphyseal vessels {Brookes, 1964).

2.3- The Microdrculation of the Bone

Long bones contain two microcirculation Systems whose interrelationship is unresolved. Some consider that the marrow and cortical microcirculation are arranged as a portai System in which arterial blood first passes through the medullary sinusoids and then perfuses the cortical capillaries. This System appears to exist in rodents (.De Bruyn et al., 1970).

The more widely accepted concept which is described below, is that the two microcirculations are distinct and separate. Lopez-Curto et al. {1980) show in their three dimensional study of the microvascular anatomy that the cortical bone and marrow capillary beds run in parallel in the dog tibia, radier than as a portai System.

There is a marked similarity between the intraosseous vascular anatomy in the long bones of the dog and the human {Nelson et al., 1960; Lopez-Curto et al.,

1980). In addition to their separate arterial input, the capillary network of the

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Figure 2.1: Diagram of the Vascular Anatomy of the Tibial Diaphysis from Lopez-Curto et al., 1980 (with permission)

NA. Nutriant artery N.V. Nutrient vain

C.M.S. Contrai medirilary statua A Artariolaa

SIN ModuHary ainuaokia

L.B. Latoral branchas of nu triant artary CAP Havarsian capliiarias

P.V. Pariosteal votai E.V. Emissary vain

2.31- The Marrow Microcirculation

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vessels. The importance of ils function is suggested by the fact thaï the marrow fat is spared during the widespread lipolysis of starvation. The capillaries of the adipose marrow are of the continuons type. However, the capillaries of the hematopoietic marrow are of the discontinuous type characterized by the observation that the sinusoids are walled by reticular cells with many fenestrations

(Kelly, J 983). The meduUary sinusoids drain into radially oriented collecting

sinuses which converge and drain into the large, central medullary sinus. Thus, the general direction of the blood flow is centripetal.

2.32- The Cortical Microdrculation

The arierioles supplying the cortex run centrifugally, from the endosteal surface, entering the cortex singly or in bundles of two to six artérioles. The cortical (Haversian) capillaries are the exchange vessels of the cortical bone. Each is widely separated from its neighbour in mature bone and is surrounded by an array of osleocytes. Cortical capillaries are long and wide compared to the capillaries of other tissue; they run in a gentle spiral relative to the long axis of the bone. They appear as a closed, continuons endothélial tube on électron microscopy. Slits or pores within the endothélial wall range in size from 3 to 4 nm for the small-pore System, to approximately 70 nm for the less numerous large- pore System (Cooper et ai, 1966). They drain into the periosteal venons System; therefore blood flows within the cortex in a centrifugal direction.

2.4- Venous Drainage

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The relative importance of the various venons channels remains controversial. Less than 10% of the blood supply of the bone and the marrow leaves by the nutrient vein {Cofield et ai, 1975), the greater proportion probably leaving by the numerous metaphyseal and epiphyseal veins (Steinbach et ai,

1957). Recent techniques using a marrow injection technique to outline the venons

System (oeteo-medullography) hâve suggested that no communications are présent between the central venons sinus and the nutrient vein (Oni et ai, 1990a). This issue remains controversial and pressure measurement studies suggest that there is a direct communication between the nutrient vein and the central medullary sinus, with the medullary pressure being higher {Wikes et al., 1975). It is likely therefore that the venons drainage from the cortical bone will preferentially flow toward the periosteal veins rather than the central medullary sinus. Flow through the central medullary sinus will be dépendent on the pressure within the bone. This may explain the fmdings of Oni et al. {1990a): if the marrow injection is not directly intravascular or the injection pressure not sufficient, then filling of the central sinus will not occur.

2.5- Lymphatics

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3- Control Mechanism of the Bone Blood Flow

3.1 Introduction

Blood flow to an organ System is regulated by the degree of vasomotor tone of small arteries and artérioles (Guyton, 1986), and each organ has its own unique physiological characteristics.

Circulation through the vascular bed of the bone is essential for maintenance of osseous homeostasis. A better knowledge of the control mechanism of bone blood flow will help to understand some of the clinical problems related to bone pathology.

Whether a bone is small or large, tubular or flat, cortical or cancellous, it is a highly vascular tissue and organ. In man, skeletal blood flow accounts for sixteen percent of the cardiac output, a figure which is comparable with rénal blood flow {Charkes, 1980). The venous capacity of bones is estimated at six - eight times that of the arterial System {Ecoiffier et ai, 1957) and therefore it raay form an important venous réservoir for the general circulation.

The physiology of blood circulation is generally concemed with the dynamic aspects of blood flow through the vascular Systems and especially with the mechanism of its control as well as the factors affecting it and their mode of action.

The rate of blood flow to the bone, as with other organs and tissue, is controlled by systemic and local control mechanisms. Systemic arterial pressure, cardiac output, and circulating blood volume affect bone blood flow with, as expected, flow declining with decreased blood pressure {Shim et ai, 1967). This is, in part mediated by baroreceptors reflexes, stimulation of which increases flow, conversely denervation decreases flow {Gross étal., 1979).

In reviewing the local mechanisms underlying the control of bone blood flow, we will subsequently consider the following:

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3 - Metabolic factors;

4 - Endothélial cell mechanisms;, with spécial emphasis on this last topic.

3.2- Neural Mechanisms

A variety of studies demonstrate the importance of sympathetic innervation for the control of bone blood flow. Sympathetic stimulation causes a decrease in bone blood flow and an increase in perfusion pressure (Drinker et ai, 1916;

Herzig et al., 1959; Azuma, 1964; Shim et ai, 1967; Driessens et ai, 1979; Tran, 1980). In contrast, a marked increase in bone blood flow has been observed after

sympathectomy {Trotman et al., 1963; Shim et al., 1972; Davis et ai, 1987) or after spinal paralysis (Verhas et al, 1980; Takahashi et al, 1990).

From the morphological standpoint, sympathetic nerve fibers hâve been identified accompanying the nutrient, metaphyseal a&^nd epiphyseal arteries. They divide with the branching of the nutrient vessels and ultimately end as multiple varicosities in a rich plexus on the adventitial surface of the small artérioles of the marrow and cortex. Fine tertiary nerves form a rich plexus on the adventitial surface of the tunica media {Sherman, 1963; Duncam et al, 1977).

These nerves contain multiple fascicles of both myelinated and unmyelinated fibers. The fmding of unmyelinated C fibers suggests an autonomie fonction {Cooper, 1968). The tertiary fibers are varicose and the sélective uptake of 5-hydroxydopamine within the vesicles of certain axonal expansions suggests the presence of the sympathetic System at this level. These probably fonction as non terminal neuromuscular synapses {Duncam et al, 1977).

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adrenergic antagonists lo abolish the constrictor effect of sympathetic nerve stimulation (Neild et ai, 1991).

Nerve fibers containing vasoactive intestinal peptide (VIP) bave been found in the periosteum and possibly the bone itself, particularly in the epiphyses. VIP is a vasodilator in most vascular beds and is often found in conjunction with acétylcholine in parasympathetic nerve endings {Lincoln et ai, 1990). Muscarinic cholinergic receptors are présent in the bone vasculature and exogenous acétylcholine causes atténuation of the responses to noradrenaline which can be inhibited by atropine, a muscarinic receptor antagonist {Woodhouse, 1963;

Driessens et ai, 1979). However, nerve fibers containing vasoactive intestinal

peptide do not appear to be associated with blood vessels {Hohmann et al., 1986;

Bjurhôlm et al, 1988) and cholinergic nerve fibers hâve not been identified as of

yet in bone. Thus, the rôle of VIP and acétylcholine is still somewhat controversial by lack of strong supporting evidence of their direct involvement in the régulation of the bone blood flow.

Other neurotransmitters with potent vasodilator effects such as substance P, an undecapeptide widely distributed within the neural tissue, and calcitonin gene-related peptide (CGRP), a 37 amino acid peptide, which is generated from the calcitonin gene by alternative RNA processing {Rosenfield et al, 1983; Neild et

al., 1991) hâve been isolated in the neural tissue of bone, marrow and periosteum {Bjurhôlm et al., 1988). However, their rôle in blood flow régulation is unknown.

The sympathetic innervation of bones appears to médiate a vasoconstrictor effect that is likely to contribute to the redistribution of the cardiac output during exercise or hypovolémie shock. Thus it causes a réduction in bone blood flow

{Gross et al., 1979). Although the intraosseous vessels are richly innervated by

sympathetic adrenergic fibers, the rank of this neural component in the hierarchy of other controlling factors is uncertain.

3.3- Humoral Factors

Circulating catecholamines such as adrénaline and noradrenaline increase bone vascular résistance and reduce bone blood flow {Drinker et ai, 1916;

Woodhouse, 1963), this effect is mediated by both and a2~ adrenergic

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Crucial to understanding the remarkably diverse effects of the catecholamines and related syrapathomimetic agents is an understanding of the classification and properties of the different types of adrenergic receptors. Ahlquist {1948) proposed the désignation a and P for receptors on smooth muscle where catecholamines produce excitatory and inhibitory responses, respectively. They are further classified by the rank order of potency of agonists. This initial classification of adrenergic receptors was corroborated by the finding that certain antagonists produce sélective blockade of the effects of adrenergic nerve impulses and sympathomimetic agents act as agonists at a-adrenergic receptors (e.g., phenoxybenzamine), whereas others produce sélective P-adrenergic blockade (e.g., propranolol). P* Receptors were later subdivided into Pi and P2, because epinephrine and norepinephrine are essentially equipotent at the former sites whereas epinephrine is 10- to 50-fold more potent than norepinephrine at the latter

{Lands et al., 1967). The stimulation of both Pi and P2 receptors are brought about by activation of adenylate cyclase, with a conséquent increase in intracellular cyclic AMP.

A human gene that encodes a third P-adrenergic receptor (designated P3) has recently been isolated {Emorine et ai, 1989) but does not seem to be involved in vascular physiology.

Heterogeneity of a-adrenergic receptors is also now widely appreciated. They are classified according to their pharmacological and functional properties. Receptors ai produce their effects by activating phospholipase C, whereas a2-receptors produce their effects by inhibiting adenylate cyclase and thus decreasing intracellular cyclic AMP. In the circulatory System, ai receptors are thought to be primarily postsynaptic and are associated with sympathetic nerve endings (van Zwieten, 1988). Receptors 0.2 are non-

innervated and regarded as hormone receptors for circulating epinephrine and norepinephrine.

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Figure 3.1: Influence of Circulating Cathechoiamine on Bone Blood Flow

This effect may possibly be explained by the presence of presynaptic P2 receptors which médiate norepinephrine release from syrapathetic nerve endings {van

Zwieten, 1988) and the absence of postsynaptic P receptors. Lack of a postsynaptic

3 mediated dilator raechanism may explain the marked constrictor effect seen in bone in response to sympathetic stimulation {Brinker et al, 1990). Apart from circulating catecholamines, other humoral factors hâve been implicated in the régulation of bone circulation.

In the isolated perfused dog tibia, it has been demonstrated that porcine calcitonin infused arterially caused vasoconstriction, an effect which is not blocked by the a-adrenergic antagonist phentolamine. Thus calcitonin may exert direct vasoconstrictor effects in bone {Driessens et al, 1981). Interestingly, this contractile response to calcitonin is seen in patients with Paget's disease {Wooton

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blood vessels in the isolated dog tibia perfusion model {Driessens et al., 1981); these results bave been confirmed in vivo by Cochrane et al .{1991).

Moreover, in the perfused tibia of the dog, low concentration of hydrocortisone augment norepinephrine-induced vasoconstriction. However, high concentrations abolish the vasoconstriction in response to nerve stimulation and norepinephrine infusion, probably by an atténuation of the vascular smooth muscle responsiveness {Driessens et al., 1981).

Estradiol has been found to depress bone blood flow in rats of both sexes. The decrease in bone blood flow after injection of estradiol benzoate shows a significant dose effect corrélation. Similarily oophorectomy increases bone blood flow {Eglise et ai, 1992). In addition to its blood flow effects, estradiol benzoate causes a similar decrease in the incorporation of "^^Ca and ^H-proline into bone, with a simultaneous increase in bone density {Kapitola et ai, 1992). A parallel increase in bone blood flow and bone loss has been noted in paraplegia {Verhas et

al., 1980) and after orchiectomy {Schoutens et al, 1984). However, in diabètes

melitus the induced bone loss is characterized by decreased bone turnover with decreased bone blood flow {Lucas, 1987). The reasons for this apparent discrepancy are not known; at présent very little is known about the rôle of the bone circulation regulatory mechanisms and some of the complex processes that affect osseous tissue.

3.4- Metabolic Factors

In many organs blood flow is controlled by the direct action of tissue hydrogen ion concentration on blood vessels {Ganong, 1989). As hydrogen ions are produced by cellular respiration, this action may synchronize blood flow with metabolic activity. The hydrogen ion concentration has a modulating effect on baseline vascular résistance and also modulâtes the response to exogenous norepinephrine ( Davis et al, 1993).

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Wray, 1988). Acidosis affects adrenergic responsiveness causing a réduction in a

adrenergic mediated effects, whereas alkalosis usually causes an increase in aj responses but atténuation of Œ2 mediated responses {Kortanje et ai, 1985).

In vivo experiments in bone demonstrate that CO2 rétention and acidosis causes vasodilation {Woodhouse, 1963; Shim SS et ai, 1967; Gross et ai, 1979). However, these observations are difficult to interpret in regard of the part played by local control mechanisms since systemic raetabolic manipulation appear to médiate the distant neural reflexes that also affect bone blood flow {Cumming et

ai, 1962).

During exercise an important factor mediating hyperemia is hyperosmolarity, presumably resulting from hyperlactosis {Mellander et ai,

1978). Potassium stimulâtes Na-K-ATPase in the cell membrane, causing

relaxation of the vascular smooth muscle and thus resulting in vasodilation

{Haddy, 1978).

The effects of hypoxia on bone blood flow hâve been investigated by different authors with contradictory results {Shim et ai, 1967; T0ndevold et ai,

1979; Gross et ai, 1979). The reason for this discrepancy in results is not readily

apparent. Hypoxia may cause vasodilation at a local level by a direct effect on the cell membrane permeability to Ca^+, causing hyperpolarization. In addition, alterations in other ion transport mechanisms, as well as interférence with electromechanical coupling and ATP production in smooth muscle may also contribute {Lombard et ai, 1988). In vascular smooth muscle 50-80% of glucose is metabolized by the glycolytic pathway to lactate under aérobic conditions, but 50-60% of the ATP production is dépendent on oxygen via the tricarboxylic acid cycle and hexosemonophosphate shunt {Lundhôlm et ai, 1977).

3.5- Endothélial Cell Mechanisms

3.51- The Concept of Endothélial Régulation of Vascular Tone

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tissues. The discovery that endothélial cells are active contributors to the balance of pro- and anti-coagulant activities {Harker, 1988) has changed this perception.

Il is now also widely recognized that the endothélium plays an important rôle in regulating vascular tone. Thus the contractility of blood vessels is regulated by varions neural and hormonal signais in concert with the local regulatory mechanisms intrinsic to the blood vessel wall. Since the discovery of prostacyclin by Moncada et al. {1979), much attention has been paid to the importance of the vasoactive substances produced within the vessel wall. Furchgott et al. (1980) demonstrated the obligatory rôle of the vascular endothélium in the vasodilatation induced by acétylcholine. This was followed by the discovery of an extremely short-lived diffusible substance which is involved in this response {Furchgott et ai, 1980). This substance has been named "Endothelium-Derived Relaxing Factor" (EDRF). In addition, a protease-sensitive vasoconstrictor peptide has also been identified in the supematant fraction of cultured bovine aortic endothélial cells {Hickey et ai, 1985; Gillespie et ai, 1986;

O'Brien et ai 1987). This substance has been isolated and purified and

subsequently synthesized by Yanagisawa et al. {1988) and termed "endothelin". These pioneer works hâve stimulated great interest in the direct rôle of the endothélium in modulating vascular responsiveness in a variety of tissues and vessels.

The production of EDRF and vasodilator prostaglandins {Davis et ai,

1992), as well as the potent vasoconstrictor effect of exogenous endothelin {Brinker et ai, 1990), hâve been demonstrated in the bone microvasculature.

Moreover, recently published works hâve implicated endothelium-produced prostaglandins, nitric oxide and endothelin in the control mechanism of bone metabolism {Alam et ai, 1992; Chambers et ai, 1991; Macintyre et ai, 1991;

Raisz, 1982; Tatrai et ai, 1992; Vaes, 1988; Zaidi et ai, 1993).

In order to hâve a better understanding of this relatively new concept of endothélium mediated régulations, the known molécules that hâve been implicated in the bone vascular physiology will subsequently be reviewed.

3.52- Vasodilator Prostaglandins

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causes vasodilation in most vascular beds (Rubanyi et ai, 1985), an effect which is dépendent on the presence of endothélial cells. This effect can be inhibited by indomethacin, a cyclooxygenase inhibitor.

Arachidonic acid is produced endogenously by the action of phospholipase A2 on membrane phospholipids. It is metabolized by three main pathways: the cyclooxygenase pathway which produces prostaglandins and thromboxanes; the lipoxygenase pathway which produces hydroxyeicosatetranoic acids (HETEs) and dihydroxyeicosatetranoic acids (di-HETEs), the function of which is uncertain, and also leukotrienes, which amongst other things are known to hâve vasodilator and vasoconstrictor properties; and the cytochrome P450 pathway which also produces HETEs and di-HETEs {Gryglewski étal., 1988; Johns étal, 1988).

In the cyclooxygenase pathway, arachidonic acid is converted to the endoperoxide PGG2 and PGH2 and then to prostacyclin (PGI2), prostaglandins PGE2, PGF2, and PGD2, and thromboxanes A2 and 82. Endothélial cells produce mainly PGI2 or PGE2 {Moncada et al, 1977)^

Prostaglandin E2 is synthesized by endothélial cells of some microcirculation {Gerritsen et al, 1983). It is known to be produced in response to angiotensin II, and tachyphylaxis to repeated doses can be inhibited by prostaglandin synthetase inhibitors (Vane et al, 1975). PGE2 production is also stimulated by bradykinin, and by circulating as well as neural induced norepinephrine. PGE2 in tum, inhibits norepinephrine release from sympathetic nerve endings.

Prostacyclin (PGI2) is another potent vasodilator prostaglandin which is produced from the vascular endothélial cell (Gerritsen et al, 1983; Moncada et

al, 1977; Weksjer et al, 1978) and is stimulated by thrombin, trypsin and the

Ca^+ ionophore Al'ilSl ( Weksjer et al, 1978). Prostacyclin appears to be the main prostaglandin product of the vascular endothélial cell {Johns et al, 1988). It has a half life of 2-3 minutes, being degraded to the inactive 6-keto-PGFjo^ which can be measured in the plasma {Moncada et al, 1978).

Prostacyclin activâtes adenylate cyclase in vascular smooth muscle cells which raises the level of cyclic adenosine monophosphate (cAMP, Gryglewski et

al, 1988). cAMP is an important modulator of cellular functions; it can activate

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cAMP dépendent protein kinases, which in tum causes phosphorylation of target proteins thus modulating their function {Kramer et al., J980). In vascular smooth muscle the effect of increased cAMP levels with regard to contractile function may be to increase binding of cytoplasmic Ca2+ to sarcoplasmic réticulum, and to phosphorylate tropomyosin. This latter substance is one of the constituents of the thin filaments of the myofibrils of smooth muscle; the thick filaments are comosed of myosin.

Figure 3.2: Metabolic Products from Arachidonic Acid

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Prostaglandins appear to be important modulators of vascular tone; it also markedly reduces postischemic reactive hyperaemia, and abolishes autoregulation in the canine kidney {Varie et al., 1975). In bone, there is some evidence that the vascular endothélium produces vasodilator protaglandins after stimulation with acétylcholine ( Davis et al., 1992).

The atténuation of vasoconstrictor responses by prostaglandins is independent of their vasodilator action. The mechanism for this is uncertain. The atténuation of noradrenaline, angiotensin, and vasopressin responses persists long after the vasodilator action of the prostaglandin has passed {Messina et al., 1976).

In addition to its rôle in vascular control, prostacyclin has potent antiplatelet actions, inhibiting platelet adhesion and aggregation {Moncada et ai,

1978; Weksjer et ai, 1977).

3.53- Endotheliiun-Derived Relædng Factor

In 1980, Furchgott et al., using isolated aortic ring préparations, demonstrated that acétylcholine can induce sraooth muscle relaxation only if endothélial cells are présent. This action is blocked by atropine and so they deducted that acétylcholine acts on endothélial muscarinic receptors to stimulate the release of a soluble smooth muscle relaxing factor. This substance was later identified as nitric oxide (NO) or a closely related nitroso compound {Moncada et

ai, 1988; Myers et al., 1990).

Further work on isolated arterial segments has confirmed that other agents require the presence of endothélium to produce smooth muscle relaxation. The release of NO may also be stimulated by shear stress on the vessel wall {Rubanyi,

1986) and may be modulated by both the frequency and amplitude of pulsatile

flow {Hutcheson et al., 1991). In addition to NO release in response to spécifie stimuli, many studies hâve also indicated on-going production of NO by the endothélium {Martin et ai, 1986; Collins et ai, 1986; Jackson, 1988). This basal EDRF release can modulate the responses of blood vessels to other vasoactive stimuli {Cocks étal., 1983; Bullock, 1986).

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lhe bone vascular bed (Davis et ai, 1991). This action bas been demonstrated in other blood vessels and is mediated by endothélial a2-adrenergic mechanisms

(Angus et ai, 1986).

NO is a labile compound, with a half-life on the order of 6 to 60 seconds

(Angus et ai, 1987; Grylewski et ai, 1986). It is avidly degraded by the oxygen-

derived free radical, superoxide anion (02') (Rubyani et ai, 1986; Moncada,

1986). The major biochemical effect of NO is activation of guanylate cyclase in

smooth muscle, resulting in accumulation of cyclic guanosine monophosphate (cGMP). This substance inhibits the contractile process by activating cGMP- dependent déphosphorylation of myosin light chains (Rapoport et ai, 1984). The decrease in myosin light-chain phosphorylation observed with cyclic GMP- mediated relaxation is most likely a conséquence of the réduction in intracellular calcium ion concentration (Rapoport, 1986).

Endothélial cells produce NO by metabolizing a terminal guanidino nitrogen atom from L-arginine. The process is stereospecific and can be inhibited by a variety of analogues of L-arginine including N^-monomethyl-L-arginine (L- NMMA). This effect is slow to disappear, unless accelerated by the addition of exogenous L-arginine (Rees et ai, 1989).

It is now recognized that EDRF coordinates the behaviour of vascular résistance vessels (Griffith et ai, 1987). In addition, it is a potent inhibitor of platelet aggregation, acting synergistically with prostacyclin (PGI2). Hence, at the interface between blood and blood vessel wall, EDRF may contribute to the anticoagulant properties of the endothélium (Vanhoutte, 1988). The relative importance, and basal production of EDRF varies among arteries and between arteries and veins (Vanhoutte, 1987). This may influence the incidence of intravascular clotting and may be important when selecting blood vessels for vascular grafting (Lüscher et ai, 1988).

Experimental studies hâve established that loss of the capacity of endothélial cells to produce EDRF, or loss of the ability of vascular smooth muscle cells to respond to EDRF, increases the reactivity of blood vessels to vasoconstrictor stimuli (Young et ai, 1986; Collins et ai, 1986; Cocks et ai,

1983; Carrier et ai, 1984). This has led to the hypothesis that damage to the

endothélium may precipitate vasospasm (Vanhoutte et ai, 1985; Vanhoutte,

1986). In the presence of a functional endothélium, EDRF produced in response

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such as serotonin and thromboxane, and maintain vascular patency. In the absence of the actions of EDRF, the balance is shifted toward contraction and possibly vasospasm {Vanhoutte, 1988). From these observations it is tempting to speculate that some of the vascular pathologies and dérangements of control of the cardiovascular System such as atheroma {Friedman et ai, 1986), hypertension

{Lüscher et al., 1986; Van de Voorde et al., 1986), atherosclerosis {Forstermann, 1988), and hyperlipidemia {Aksulu et al., 1986), are associated with localized

impairment of the release or effect of EDRF. Excess production of EDRF might also lead to disease. Septic shock induces the expression of a second distinct "nitric oxide synthase" enzyme in the endothélial cells {Radamski et al, 1990). Increased production of EDRF by this enzyme raay to contribute to hypotension

{Ress et al., 1990; Julou-Schaejfer et ai, 1990). It is not known if local infection

(e.g. osteomyelitis) causes induction of this enzyme.

3.54- Endothelin

Endothelin (ET) is a family of paracrine hormones with strong vasoactive properties. They are présent in at least three isoforms (ET-1, ET-2, and ET-3). Sequence analysis of cloned cDNAs for porcine {Yanagisawa et al., 1988) and human {Harker, 1988) endothelin precursors show that endothelin-1 (ET-1) is produced in the endothélial cells from a 203 amino acid precursor peptide called preproendothelin {Yanagisawa et ai, 1988). Following translation it is cleaved to form "big endothelin", a 39 amino acid peptide. A metalloprotease endothelin- converting enzyme, présent in vascular endothélial cells, then cleaves proendothelin by a unique splitting between a tryptamine-21 and a valine-22 to produce the final 21 amino acid peptide {McMahon étal, 1991).

Genomes for three different endothelins hâve been identified in the human

(Inoue et al, 7959).They hâve very similar peptide structure consisting of 21

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3.541-Presser Actions

The peptide endothelin (ET) was first identified as a potent vasoconstrictor produced by endothélial cells. It is one of the most potent endogenous vascular smooth muscle constrictors, being 10 times more potent than angiotensin II, vasopressin and neuropeptide Y {Yanagisawa étal., 1988). The prominent feature of the presser phase of the response to ET in vivo is its time course; more than 2-3 hours are typically required for retum of arterial pressure to base-line levels after a 2nmol/kg intravenons bolus in the rat {Yanagisawa et al., 1989). ET elicits a slow-developing, and long-lasting vasoconstriction in almost ail arteries and veins, but the ability of the endothélium to modulate contractions to endothelin differs between the arteries and the veins. In the relatively large vessels, veins are more sensitive than arteries to ET {Miller et al., 1989; Cocks et al., 1989). In microvessels however, artérioles appear to be more sensitive to ET than venules

{Brain, 1989).

Shortly after the discovery of this vasoconstrictor peptide, it was leamed that endothelin also possesses vasodilator properties at doses lower than those necessary to produce vasoconstriction {Baydoun et al., 1990; Warner et al., 1989;

Gardiner et al, 1989). However, controversy exists over the mechanism(s) of

action; prostacyclin and endothélium derived relaxing factor (EDRF) hâve mainly been implicated as the source of the initial vasodepressor effect {de Nucci et al,

1988; Warner et al, 1989; Herman et al, 1989). ET also elicits markedly

different régional hémodynamie response patterns {Le Monnier de Gouville et al,

1990). There is a heterogeneity in the observed vasodilation or vasoconstriction

actions dépendent on species and on the studied vascular beds in the same species

{Egden et al, 1989; Tsuchiya et al, 1989; Wright et al, 1988). This observation

may be related to différences in the direct response to endothelin or to its modulation by other agents such as prostaglandins, EDRF, or adrenergic mechanisms.

The three ETs possess a common spectrum of biological activities, i.e. vasodilation and vasoconstriction, but their biological potencies differ {de Nucci et

al, 1988; Inoue et al, 1989; Spokes et al, 1989). ET-2 is slightly less potent but

has a greater duration of its contractile response than endothelin-1 and endothelin- 3 is the least potent as a vasoconstrictor {Inoue et al, 1989;Yanagisawa et al,

1988).

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response {Inoue et ai, 1989;Yanagisawa et al., 1988). The initial depressor response is thought to be endothélium dépendent. Regarding the initial depressor activity, ET-3 is the most potent of the three.

3.542- Endothelin Receptors in the Vascular Tissue

The differential potencies of ET-1 and ET-3 suggests that subtypes of the endothelin receptor exist (Masaki et al., 1991). Indeed, two subtypes hâve been cloned and sequenced (Aral et al., 1990; Sakurai et al., 1990). They are denoted ET-A and ET-B.

Table 3.1: Classiffîcation of Endothelin Receptors on the Vascular Tissue

Typje of Receptor Affinity Rank Order Localization Effect

ET-A ET-1 > ET-3 smooth muscle vasoconstriction

ETB ET-1 = ET-3 endothélium vasodilation: release of

EDRF / prostacyclin

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ET-A shows a high specificity for endothelin- 1 and has been found on the vascular smooth muscle cells {Hirata et al, 1988; Lin et ai, 1991). ET-B, less spécifie shares equal affînity for the different endothelins and has been localized in the vascular endothélial (Takayannagi et ai, 1991) and smooth muscle cells

{Shetty et ai, 1993). The ET-B receptors located on the endothélium are thought

to médiate the vasodilator action of endothelin (Shetty et ai, 1993; Sakurai et ai,

1992).

Studies involving synthetic antagonists (BQ -123, and BQ -153) for the ETA receptor (Ihara et ai, 1991; Ihara et al, 1992; Fukuroda et ai, 1992) confirm that the ETA receptor médiates most of the vascular smooth muscle contraction, but a small and variable proportion of the contractile response to endothelin is résistant to those agents indicating that it is in part mediated by another receptor located on the smooth muscle (Fukuroda et ai, 1992).

Figure 3.3: Intracellular Mechanisms et ET-1 Mediated Vasoconstriction

voltage operated receptor operateO llc> <3 protêt n

Pl-iosprioticdvi Irtosttol Oipl~»ospt^atd

2

I Inositol 1 .•4^,0-tripnospMsTe

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The intracellular mechanisras of endothelin hâve been recently reviewed

(Simonson et ai, 1992; Highsmith et al., 1992). The ETA receptors belong to the

G-protein-coupled receptor class (adrenergic and muscarinic receptors also belong to this class). One of the characteristics of stimulated G-proteins is that they may interact with other different G-proteins {Simonson et al., 1992) and therefore lead to complex cellular responses which are individualized for spécifie cellular functions.

ET-induced vasoconstriction may be mediated through two distinct intracellular signal transduction Systems; one is phospholipase C activation

{Resnik et al, 1988; Takuwa et al, 1990), the other is the opening of calcium

channels (Goto et al, 1989; Kasuya et al, 1989); both pathways require G- proteins. The first System stimulâtes phospholipase C to produce inositol 1,4,5- triphosphate (IP3) and diacylglycérol {Muldoon et al, 1989). Ip3 is soluble in the cytoplasm and binds to receptors on the endoplasmic réticulum causing release of intracellular stores of calcium {Berridge et al, 1990). Diacylglycérol diffuses through the plasma membrane and in the presence of calcium ions and phosphatidylserine activâtes protein kinase C. Activated protein kinase C, along with calmodulin protein kinases which are activated by the increased intracellular calcium levels, cause the phosphorylation of myosin light chains leading to contractile cross-linkages with actin.

Activated protein kinase C, along with calmodulin protein kinases which are activated by the increased intracellular calcium levels, cause the phosphorylation of myosin light chains leading to contractile cross-linkages with actin.

The second endothelin G-protein System directly activâtes calcium channels. There are two main types of calcium channels {Resnik et al, 1988) - one is the voltage operated (dihydropyridine sensitive) channel where calcium entry may be blocked by nifedipine, nicardipine, verapamil etc., and the other is the receptor operated channel. In some tissues, the effects of endothelin can be antagonized by nifedipine or verapamil {Yanagisawa et al, 1988; Kasua et al,

1989; Chen et al, 1993) while unaffected in others {Resnik et al, 1988).

Endothelin may activate both types of channel {Resnik et al, 1988; Blackbum et

al, 1990). Moreover, endothelin potentiates the effects of other agonists on

vascular smooth muscle {Goto et al, 1989, Inoue et al, 1990). This effect may be the resuit of partial membrane depolarization thus decreasing the threshold for activation of voltage operated channels, or to an alteration in the sensitivity of intracellular mechanisms {Resnik et al, 1988). It is probable that endothelin has other intracellular signaling mechanisms {Resnik et al, 1988, Takuwa et al, 1990;

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The mechanism of the vasodilator action of endothelin is uncertain. EDRF appears to be the mechanism of its dilaior action in some vascular beds {Warner et

al., 1989), and vasodilator prostaglandins in others, an action which is probably

mediated by the ETE receptor. Endothélium raay stimulate other phospholipases such as phospholipase D, and other ion channels such as the Na'*’/H‘*' exchange

{Simonson et al., 1992).

3.543- Rôle of Endothelin

Although détectable in the plasma at levels of 0.5 - 5.0 pmol/1 in the human {Ando et al., 1989: Cemacek et al., 1989; Nakamura et ai, 1990; Wagner

et al., 1990), these levels of endothelin are generally below the concentrations

required to activate the ETA receptors. This observation suggests that endothelin derived from the endothélial cell primarily acts at a local level rather than as a circulating hormone. Delay between stimulus and de novo synthesis of endothelin suggests that it may be involved in long tenu homeostasis.

Endothelin is rapidly cleared from the blood with 60% being removed by one pass through the lungs (de Nucci et ai, 198S). It is also removed in the liver and the kidney (de Nucci et ai, 1988; Ânggard et al., 1989) and in the splenic and skeletal muscle vascular beds {Pemow et al., 1989). Its half-Iife in the plasma is less than 2 minutes, but whether this its catabolism or receptor binding is uncertain.

ET probably plays an important rôle in the maintenance of blood pressure. Significantly higher plasma ET-1 levels hâve been found in patients with malignant hemangioendothelioma and concomitant hypertension, in patients with essential hypertension, and in hemodialized hypertensive patients {Yokokawa et

al., 1991; Saito et ai, 1990; Shichiri et al., 1990). Moreover, ET appears to

promote smooth muscle prolifération which may also contribute to increased vascular résistance {Komuro et ai, 1988). However, the plasma ET level associated with essential hypertension is still the subject of controversy.

Endothelin has been implicated in a number of pathological situations including tissue ischemia {Vemulapalli et ai, 1992; Watanabe et al, 1991;

Mualik et al., 1992) and vasospasm. Numerous studies hâve shown that there is

an increase in the endothelin receptor density or increased affmity for endothelin after ischemia in the heart or kidneys {Liu et al., 1990; Clozel et ai, 1991; Watts

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effective in reducing infarct size in üie heart (Liu et al., 1990), and in restoring near normal vascular résistances in the rénal vascular bed after ischeraia (ATon et

al., 1991). ET-l,synthesized in dysfunctional endothélium can cause secondary

vasospasm. Indeed, actinomycin D, a transcriptional inhibitor, completely inhibits the development of the chronic vasospasm that was observed in ail nontreated Controls 1 week after subarachnoid hemorrhage in the rat (Shigeno et al., 1991).

The basal expression of the mRNA for the preproendothelin in the cultured porcine aortic endothélial cells seems to be much higher than that in the aortic endothélial cells in vivo. It has been generally accepted that cultured endothélial cells approximate the functional State of injured endothélium in vivo {Ross, 1986). Furthermore, thrombin and increased shear stress are known stimulators of the expression of ET. It can therefore be speculated that ET is produced more actively around the site of endothélial damage. In such instances, the balance between the vasodilator and vasoconstrictor induced responses may be lost which may contribute to the pathogenesis of vasospasm.

Experimental results strongly support the concept that ET is important physiologically and pathophysiologically in controlling vascular tonus {Masaki et

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4- Vascularized Bone Grafts

4.1- Development of Vascularized Bone Grafts

The earliest record of autogenous pedicled grafts is in the sanskrit text of India, the Sushrista Shamita {Flye MW, 1989). These grafts were used to reconstruct mutilations of the face in the second century BC. The modem concept of transplanting a vascularized, living bone was proposed by Phelps {1891) and Curtis (1893), and the first transfer of the ipsilateral fibuia onto the tibia, was described by Huntington in 1905 (Huntington, 1905). However, the use of such pedicled grafts is limited by the length of the vascular pedicle. In most anatomical sites, vascular bone transplantation did not become practical until the operating microscope, and improved microsurgical instmmentation became available. It enabled microsurgical re-anastomosis of the vascular pedicle to the chosen récipient site (Ostrup et ai, 1974).

The first report of a free vascularized fibular graft was performed by Taylor in 1975 (Taylor et ai, 1975) and since then applied in numerous cünical situations including head and neck reconstructions (Wei et al., 1986; Hidalgo,

1989).

4.2- Healing Mechanism of Non-Vascularized Bone Grafts

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Bone incorporation requires the invasion of vascular buds and, in cortical bone, this usually occurs through pre-existing Haversian or Volkmann canals

{Shqffer et ai, 1985). This process is efficient in cancellons bone grafts because

they provide an open structure which permits rapid revascularization and more complété incorporation. In cortical bone grafts, bone must be replaced by reactive woven bone before remodeling. Significant amounts of non-viable interstitial lamellae persist in massive cortical grafts, and incorporation of the graft is usually incomplète {Enneking étal., 1975; Weiland et al, 1984).

4.3- Healing and Remodeling of Vascularized Bone Grafts

The major objective of a vascularized bone transfer is the immédiate restoration of a physiological blood supply so that the cellular éléments of the graft can survive. With an intact blood supply and viable ostéogénie cells, incorporation of a vascularized bone graft should be rapid and resemble fracture healing. Experimental studies, however, hâve demonstrated that significant bone résorption may occur in these grafts (Weiland et al., 1984; Schaffer et ai, 1987;

Goldberg et al, 1987). This is due to remodelling which progresses rapidly along

the intracortical vascular System. There is an increase in bone porosity but the architecture of the cortex is preserved {Schwarzenbach, 1989). Histomorphometric studies hâve confirmed that vascularized grafts maintain a greater cross-sectional area of cortical bone (Goldberg et ai, 1987) and peripheral new bone déposition is much less marked than in avascular autografts, where cortical bone is replaced by woven reactive bone.

Different studies hâve investigated the viability of cells within a vascularized bone graft. The number of viable cells is usually estimated by fluorochrome analysis. These dyes are taken up only by osteons that are actively involved in the process of bone formation. Because this process is variable (Tarn,

1974), the quantity of fluorochrome uptake may not be a direct measure of ail the

Figure

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