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Kaderly, Robert Elton

POSTNATAL MATURATION OF THE MICROCIRCULATION IN THE FEMUR OF THE DOG

The Ohio State University PH.D. 1984

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University Microfilms International POSTNATAL MATURATION OF THE MICROCIRCULATION

IN THE FEMUR OF THE DOG

DISSERTATION

Presented in Partial Fulfillm ent of the Requirements for

the Degree Doctor o f Philosophy in the Graduate

School o f The Ohio State U niversity

By Robert Elton Kaderly, B.S., D.V.M., M.S.

*****

The Ohio State U niversity

1984

Reading Committee: Approved By

Bettina G. Anderson, Adviser R. Bruce Hohn, Adviser Wesley D. Anderson Steven E. Weisbrode Adviser Department of Veterinary Anatomy

L • Adviser Department of Veterinary Clinical Sciences Copyright by Robert Elton Kaderly 1984 ACKNOWLEDGMENTS

I wish to thank my advisers, Bettina Anderson and Bruce Hohn for the ir assistance, encouragement, and guidance, and the other members of n\y Reading Committee, Wesley Anderson and Steven Weisbrode, fo r the time they gave from th e ir busy schedules.

I esp ecially thank Bud Kramer fo r his fin e artw ork, Linda S te ttle r and Marian M iller for their prompt photographic processing and printing, and Susan Warner for her efficient typing. Their extra e ffo rts were g re a tly appreciated.

I personally wish to thank my wife, Theresa, and daughters, Annie and Erin, for their patience, and n\y father, Elton, for his support and valuable advice. VITA

November 1 , 1942 ...... Born - Columbus, Ohio

1965 ...... B.S., Otterbein College, Westerville, Ohio

1969 ...... D.V.M., The Ohio State U n ive rsity, Columbus, Ohio

1969-1977 ...... Veterinarian, Kaderly Veterinary Clinic, Twinsburg, Ohio

1978-1979 ...... Graduate Teaching Assistant, Department of Veterinary Anatomy, College of Veterinary Medicine, The Ohio State U n ive rsity, Columbus, Ohio

1980 ...... M .Sc., The Ohio State U n ive rsity, Columbus, Ohio

1979-1982 ...... C lin ic a l In s tru c to r, Small Animal Surgery Residency, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio

1981-1984 ...... Instructor, Departments of Veterinary C lin ic a l Sciences and Veterinary Anatomy, College o f V eterinary Medicine, The Ohio State U n iv e rs ity , Columbus, Ohio

PUBLICATIONS

"Extraosseous Vascular Supply to the Mature Dog's Coxofemoral Joint." Am. J . Vet. Res., 43:1208-1214, 1982.

"Intracapsular and Intraosseous Vascular Supply to the Mature Dog's Coxofemoral Joint." Am. J. Vet. Res., 44:1805-1812, 1983.

FIELDS OF STUDY

Major Fields: Veterinary Anatomy Small Animal Surgery i i i Studies in Vascular Anatomy of Bone Professor Bettina G. Anderson

Studies in Small Animal Orthopedic Surgery Professor R. Bruce Hohn TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... i i

VITA ...... i i i

LIST OF FIGURES...... vi

PREFACE ...... 1

CHAPTER

I . MATURATION OF THE MICROCIRCULATION IN THE MARROW

Abstract ...... 5 Introduction ...... 7 M aterials and Methods ...... 8 R e s u lts...... 12 D is c u s s io n...... 79

I I . MATURATION OF THE MICROCIRCULATION IN CORTICAL BONE

A b s t r a c t ...... 89 Introduction ...... 90 M aterials and Methods ...... 92 Resul t s ...... 93 D is c u s s io n...... 113

I I I . COMMUNICATION OF THE MARROW AND CORTICAL BONE MICROCIRCULATION

A b s t r a c t ...... 122 Introduction ...... 123 M aterials and Methods ...... 124 R e s u lts...... 125 D is c u s s io n...... 138

LIST OF REFERENCES...... 145

v LIST OF FIGURES

0

CHAPTER I

Figure Page

1.1 Comparative sizes and configurations of the femur in the newborn puppy, two-week-old puppy, ten-week-old puppy, and adult dog ...... 13

1.2 Caudal one-half, proximal le ft femur (cut surface); adult dog. Arterial vascular pattern down to and including marrow sinusoids ...... 15

1.3 Caudal one-half, diaphysis of le ft femur (cut surface); adult dog. Branches of the descending medullary arteries coursing distally below the nutrient foramen ...... 18

1.4 Medial surface, shaft of le ft femur, newborn puppy. Fine arterial branches from the ascending and descending medullary arteries and the basic venous system of med­ ullary drainage ...... 21

1.5 Marrow cavity and lateral cortex (cut surface) at the level of the nutrient artery; adult dog. Nutrient artery occupying nearly the entire diameter of the nutrient canal ...... 23

1.6 Venous structures surrounding the transparent nutrient artery within the nutrient canal; adult dog ...... 23

1.7 Craniolateral edge of (laterally sectioned) proximal le ft femur, two-week-old puppy. Cranial and caudal proximal metaphyseal veins draining the proximal medullary cavity and anastomosing with the central medullary vein . . 25

1.8 Caudolateral edge of (laterally sectioned) distal right femur; two-week-old puppy. Distal metaphyseal vein passing obtusely through the cortex and draining the distal medullary ca vity ...... 25

1.9 Drawing of the diaphyseal, proximal and distal metaphyseal veins, draining the central medullary vein; adult dog . . . 28

vi Figure Page

1.10 Lateral (cut) surface of distal metaphysis, right femur; newborn puppy. Fine branches from descending medullary arteries supplying sinusoids just proximal to the distal p h y s is ...... 30

1.11 Lateral (cut) surface of distal metaphysis, le ft femur; newborn puppy. Distal metaphyseal sinusoids converging on collecting sinuses and draining through the caudal cortex via a distal metaphyseal v e in ...... 30

1.12 SEM o f marrow c e lls and trabecular bone; newborn puppy. Comparison of a la rg e , smooth-walled a rte ry and an irregular sinusoid ...... 32

1.13 SEM OF tra becu lar bone and marrow tis s u e ; newborn puppy. Developing sinusoids and collecting sinuses ...... 34

1.14 SEM of decalcified trabecular bone, marrow tissue, sinusoids, and c o lle c tin g sinuses; a d u lt dog. Several collecting sinuses converge on a large collecting vein . . 34

1.15 Arterial vessels of the distal diaphyseal marrow; adult dog. Long side branches of a large medullary artery continuing to divide into narrow arteriolar capillaries to supply well-defined sinusoids ...... 37

1.16 Arterial supply to the sinusoids through very narrow arteriolar capillaries; adult dog ...... 37

1.17 SEM of bone marrow; newborn puppy. Large artery lying adja­ cent to trabecular bone (and near a collecting vein) giv­ ing off acute, narrow side branches to supply sinusoids . . 39

1.18 SEM of bone marrow; newborn puppy. Endothelial nuclear protrusions into the lumen of the artery have created impressions in the M icrofilr material ...... 39

1.19 SEM o f a branched c o lle c tin g sinus w ith in marrow tis s u e ; newborn puppy ...... 41

1.20 SEM of migration pores within the walls of a collecting sinus; newborn puppy ...... 41

1.21 SEM o f a sinusoidal w a ll; newborn puppy. Erythrocytes adhering to sinusoidal wall and passing through migra­ tio n p o r e s...... 44

1.22 SEM of the transmural migration of marrow erythrocytes (specimen has been ro ta te d ); newborn puppy ...... 44 vii Figure Page

1.23 Vascular pattern of fatty marrow, mid-diaphysis; adult dog. Numerous venules drain patchy capillary beds .... 46

1.24 Capillary beds of fatty marrow; adult dog. Several collecting veins receive venules from capillary beds . . . 46

1.25 SEM o f a small c a p illa ry-ve n u le bed in marrow c a v ity ; newborn puppy ...... 49

1.26 SEM of anastomotic, three-dimensional venule pattern; newborn puppy ...... 49

1.27 Sinusoids draining directly into central medullary vein via long and short drainage venules; two-week-old puppy . . 51

1.28 Transcortical view of (intersinusoidal) communicating venules between several sinusoids; two-week-old puppy . . . 51

1.29 SEM of trabecular bone; newborn puppy. A long, thin (intersinusoidal) communicating venule joining two sinusoids. Developing sinusoids draining into a collecting sin u s...... 53

1.30 SEM of collecting sinus receiving a branched sinusoid; newborn puppy ...... 53

1.31 Venous structures within the medullary cavity; adult dog. Several venule beds entering collecting veins beneath the cortex at the level of the nutrient canal...... 55

1.32 A large tu ft of venules joined to a smaller venule tu ft by a single communicating venule; adult dog ...... 57

1.33 A single dimpled sinusoid, with venules leading from the sinusoid to the central medullary vein and a venule- capillary bed; adult d o g ...... 57

1.34 Craniolateral surface, le ft femoral head and neck; new­ born puppy. Vessels on the surface of the femoral neck penetrating the cartilaginous femoral head to form random epiphyseal vascular loops ...... 60

1.35 Branching vascular loops within the cartilaginous femoral head; newborn puppy ...... 60

1.36 Epiphyseal vascular loops of the cartilaginous femoral condyle; newborn puppy. Perichondrial vessels from the lateral and medial surfaces at the trochlear margin penetrating the distal epiphyseal cartilage ...... 62

vi i i Figure Page

1.37 Lateral (cut) surface, distal femoral epiphysis; new­ born puppy. Random distribution of vascular loops within the cartilaginous femoral condyle ...... 64

1.38 Lateral (cut) surface, distal femoral physis; newborn puppy. Vessels from the ends of the epiphyseal vascular loops traversing the physis and communicating with distal metaphyseal sinusoids ...... 64

1.39 SEM of the proximal physis; newborn puppy. A large vein coursing from the proximal metaphysis, across the physis and through the proximal epiphysis ...... 66

1.40 Cranial surface, right femoral head and neck; two-week-old puppy. Vascular supply to , and venous drainage from, the secondary center of ossification ...... 68

1.41 Subsynovial veins on the cranial surface of the femoral neck draining the secondary center of ossification; two- week-old puppy ...... 68

1.42 Lateral (cut) surface of distal femur; ten-week-old puppy. Developing secondary ossification center, incorporating more and more epiphyseal cartilage and giving better definition to the physis and articular cartilage ...... 71

1.43 Vessels of the secondary ossification center; ten-week-old puppy. Residual vascular loops within the articular carti­ lage are continuous with the vessels of the expanding secondary ossification center across a plate of sub­ chondral bone ...... 71

1.44 Caudal articular surface, femoral head; adult dog. Subchondral vascular c o n tin u ity between a r te ria l c a p il­ laries, sinusoids and collecting sinuses ...... 73

1.45 Vascular loops within the ligament to the head of the femur; adult dog. Ligament vessels are blocked from epiphyseal vessels by a plate of articular cartilage . . . 73

1.46 Drawing o f the a r te ria l and venous marrow m icro­ c irc u la tio n ; newborn puppy ...... 75

1.47 Drawing of the microvascular relationships at the proximal and distal physes; ten-week-old puppy ...... 77

ix CHAPTER I I

Figure Page

2.1 Cross-section of mid-diaphysis; adult dog. Cortical vessels radiating outwardly from the endosteal surface to the periosteum...... 95

2.2 Cross-section of cranial cortex, mid-diaphysis; adult dog. Demarcation of outer and inner circumferential lamellae from middle osteonal layer ...... 95

2.3 Inner cortex and outer marrow c a v ity (cu t surface); adult dog. Multiple arterioles entering a single pene­ trating canal at the endosteal surface ...... 97

2.4 Inner cortex and outer marrow cavity (cut surface); adult dog. Tortuosity of arteriolar branches at the endosteal s u rfa c e ...... 97

2.5 Venous structures within the diaphyseal cortex; ten-week- old puppy. Endosteal sinusoids giving rise to portal venules which traverse the cortex to the deep periosteum . 99

2.6 Diaphyseal cortex (cut surface); ten-week-old puppy. Cortical venules joining the deep periosteal plexus.... 99

2.7 Cortex and endosteum (cu t su rfa ce ); a d u lt dog. Portal venules from endosteal sinusoids giving off longitudinally oriented side branches as they travel outwardly through the c o r t e x...... 102

2.8 Cortex, endosteum, and outer medullary c a v ity ; a d u lt dog. Cortical arterioles and venules communicating between the deep periosteum and the marrow c a v it y ...... 102

2.9 Cortex, endosteum, and outer medullary cavity (cut surface); adult dog. Evidence of cortical remodeling by vessels within several resorption cavities ...... 104

2.10 Cortex (cut surface); adult dog. Tuft of vessels at the "cutter cone" head of a resorption cavity ...... 106

2.11 Cortex (cu t surface); a d u lt dog. A rte rio le b ifu rc a tin g a t the apex o f a resorption ca vity and forming two re ­ turning venules...... 106

2.12 Lateral (cut) surface, right femoral diaphysis; newborn puppy. Cortical vessels emanating from mid-diaphyseal fo c u s...... 109

x Figure Page

2.13 Periosteal surface (above) and cut cortical surface (below), mid-diaphysis; newborn puppy. Cortical vessels draining into the deep periosteal p le xu s...... 109

2.14 Periosteal surface, mid-diaphysis; ten-week-old puppy. Superficial periosteal vessels, deep periosteal plexus, and endosteal vessels ...... I l l

2.15 Periosteal surface, mid-diaphysis; adult dog. Super­ ficia l periosteal vessels originating from the linea a s p e r a ...... I l l

2.16 Drawing o f the a rte ria l and venous m ic ro c irc u la tio n in woven c o rtic a l bone; newborn puppy ...... 114

CHAPTER I I I

3.1 Endosteal surface, mid-diaphysis; adult dog. Small arteriole supplying endosteal sinusoid from which a portal venule arises ...... 126

3.2 Endosteal surface, mid-diaphysis; adult dog. Collect­ ing sinuses and portal venules draining endosteal s in u s o id s ...... 126

3.3 Transcortical venous drainage of a large capillary- venule bed at the periphery of the medullary cavity; a d u lt d o g ...... 129

3.4 Venules leaving the capillary-venule bed and entering the endosteal cortical surface; adult dog ...... 129

3.5 Cortex and endosteum (cu t su rfa ce ); a d u lt dog. A re­ sorption cavity being supplied by a cortical arteriole and drained by a cortical venule to the periosteum .... 131

3.6 Cortex (cut surface); adult dog. Medullary supply and periosteal drainage of a resorption cavity ...... 131

3.7 Cortex and endosteum (cu t surface); a d u lt dog. A re­ sorption cavity being supplied by a cortical arteriole and drained by a venule of an endosteal sinusoid ...... 134

3.8 Cortex and endosteum (cu t su rfa ce ); a d u lt dog. A medullary arteriole entering the cortex, branching and returning to the medullary cavity to supply an endosteal sinusoid . . . 134

xi Figure Page

3.9 Cortex (cut surface); adult dog. Venules of an intracortical capiliary-venule anastomosis draining directly into the central medullary v e in ...... 136

3.10 Cortex (cut surface); adult dog. Isolated sinusoids entrapped within cortical bone ...... 136

3.11 Schematic drawing of unidirectional and bi-directional vascular intercommunications between marrow and cortical bone microcirculatory systems ...... 139

x ii PREFACE

The microcirculation of long bones may be a rtific ia lly subdivided in to (1) a rte ria l supply and venous drainage o f the marrow, and ( i i ) a r te ria l supply and venous drainage o f c o rtic a l bone. The m icrovascular systems o f marrow and c o rtic a l bone are not m utually exclusive. One re la tio n s h ip between the two systems is obvious: extraosseous arteries must traverse cortical bone through vascular channels to supply the marrow, and venous structures must leave the medullary cavity through vascular channels within the cortex to enter the extraosseous venous system.

Although the subject of venous drainage of long bones has not received as much investigative attention as arterial supply, maintaining adequate venous drainage could be much more important than arterial supply for normal microcirculation. The unyielding diameter of vascular channels passing through rigid cortical bone places a unique re s tric tio n on the capacity o f venous stru cture s to compensate for increased afferent flow. Impaired blood flow through the marrow would inhibit the entry of blood cells, platelets and tissue fluid into the systemic circulation, while also compromising the nutrition of the marrow tissue and cancellous bone. Poor venous drainage of bone may predispose to osteonecrosis1* 2 and possibly delay or prevent fracture healing in some instances.3 Stagnation of blood due to obstructed venous outflow may be involved in the establishment of osteomyelitis during bacteremic episodes,^ the sequential development of bone 5 sarcomas in m u ltifo ca l areas o f bone in fa rc tio n , hematogenous metastasis o f various neoplasms to bony s ite s , the pathogenesis o f aneurysmal bone cysts,^ or the necrosis of supporting cancellous bone 8-10 in conditions such as Legg-Calve-Perthes' disease.

Venous drainage patterns and directional blood flow from marrow 11 and cortical bone remains a controversial issue. The difficulties of studying minute vessels encased within an opaque, mineralized shell are compounded when attempting to separate the microcirculatory vascular stru cture s in to a rte ria l and venous components. S e le ctive ly profusing only venous structures is te c h n ic a lly d if f ic u lt . Complete f i l l i n g o f a ll intraosseous vessels with a common material makes the d iffe re n tia tio n o f a rte ria l and venous vessels unclear. In a d d itio n , the morphological characteristics of static vascular structures do not always suggest the direction of blood flow. Various other techniques

(bone blood flow measurements, vascular pressure recordings) can be helpful in interpreting anatomical information.

The following investigation was designed to further describe the microcirculatory structures and vascular patterns in marrow and cortical bone. Structural evidence supporting the hypothesis of centrifugal and centripetal transcortical blood flow was sought. By following to maturity the relatively simplified arterial and venous systems present in the newborn puppy, the complex microcirculatory patterns in the adult dog could be interpreted more accurately. An emphasis was placed on in ve s tig a tin g venous stru cture s and patterns because o f the p o ten tial c lin ic a l relevance o f impaired venous drainage and the current lack of a definitive study of the venous drainage of long bones. Attention was concentrated in three areas: (i) maturation of the marrow microcirculation, (ii) maturation of the microcirculation in c o rtic a l bone, and ( i i i ) the communications of marrow and c o rtic a l bone microcirculation. CHAPTER I

MATURATION OF THE MICROCIRCULATION IN THE MARROW

4 ABSTRACT

Proximal and distal epiphyseal, metaphyseal, and diaphyseal nut­ rient arteries entered the medullary cavity at their respective levels. Arteries within the medullary cavity branched to eventually form small arteries and arterioles. Small arteries gave rise to arteriolar capillaries which were characteristic in their acute angles of origin, abrupt narrowness, and thin walls. Arteriolar capillaries supplied sinusoids in areas of hematopoietic marrow. Arterioles terminated into capillaries which established capillary-venule beds in patchy areas of fatty marrow.

Sinusoids joined with other sinusoids through communicating ven­ ules, and emptied directly into the central medullary vein via drainage venules or indirectly into the central medullary vein via collecting sinuses and collecting veins. Transmural migration of marrow blood cells was demonstrated through migration pores in the sinusoids and collecting sinuses. The central medullary vein was (consistently) drained by cranial and caudal proximal metaphyseal and distal metaphyseal emissary veins and (inconsistently) by proximal and distal diaphyseal emissary veins.

Maturation of the microcirculation in the proximal and distal epiphyses was followed from the random cartilage vessels of newborn puppies, through the establishment and development of the microcirculation in secondary ossificiation centers of 2 and 10-week-old puppies, to obliteration of the physis and the joining of epiphyseal and metaphyseal microvacular systems of adult dogs.

Transphyseal vessels were only demonstrated in the femurs of newborn puppies. Throughout femoral development, vessels in the ligament to the head of the femur did not contribute to the microcirculation in the femoral head. 7

INTRODUCTION

Unique vascular structures o f marrow have been known to e x is t fo r many years. In 1901, Minot coined the term "sinusoid," and described

its characteristic features by comparing it to a capillary.* Vascular

d ifferences between hematopoietic and fa tty marrow were recognized

grossly by Trueta in 1953.^ Only recently, with the advent of

transmission and scanning electron microscopic techniques, has

ultrastructural detail revealed specific structural adaptations of the

a r te ria l and venous microvascular systems o f marrow, which apparently 3 are present to fa c ilita te the fun ction o f bone marrow as an organ.

While the ultrastructural features of vessels within marrow have

suggested c e rta in physiological mechanisms, from a la rg e r point of

view, a d e scrip tio n o f the dog's a rte ria l and venous marrow microvascular systems in complete continuity, is unavailable.

In general, arterial vessels within the medullary cavity supply both marrow and cortical bone microcirculations. More attention is devoted to Chapter I (Maturation of the Microcirculation in the Marrow) to appropriately "set the stage" for Chapters II and III. MATERIALS AND METHODS

Vascular injections were performed on puppies and dogs of mixed breeding and of either sex. Twelve newborn puppies weighing 0.45-0.60 kg (2 l i t t e r s ) , 6 two-week-old puppies weighing 1.6-1.8 kg (one litte r), 6 ten-week-old puppies weighing 4.5-5.0 kg (one litte r), and

17 adult dogs weighing 18-30 kg were used in this investigation. All subjects were anesthetized with intravenous or intracardiac injections o f pentobarbital sodium (approximately 25 mg/kg to e ffe c t). Each animal was heparinized w ith heparin sodium (200 USP u n its plus 10 u n its /k g o f body weight) and placed in dorsal recumbency.

In the puppies, a surgical approach was made to the caudal abdomen by a longitudinal incision through the linea alba, followed by a transverse abdominal incision at the cranial lim it of the in itia l longitudinal incision. The le ft and right sides of the abdominal wall were reflected laterally to locate the common ilia c vein and the external iliac artery just proximal to the internal inguinal rings.

Segments o f both the a rte ry and vein on each side were iso la te d and 2 s ix -in c h lengths o f 2-0 surgical s ilk suture were passed around each vessel. In the adult dogs, a ventral approach to the coxofemoral joint was substituted for the abdominal celiotomy. The medial circumflex femoral artery (source vessel of the nutrient artery to the femur) and the femoral vein were isolated after removing the overlying pectineus muscle. Two lengths of 2-0 surgical silk suture were placed around each vessels. The common ilia c vein in the puppies or the femoral vein in the adult dogs was ligated firs t, by tying the most proximal length of suture. Polyethylene male urinary catheters (sizes 3 1/2, 5, 8, or 9

10 Fr.) were incised at a 30° angle, and the distal length of suture was retracted gently to occlude the lumen of the vein. A small p e rfo ra tio n was made through the wall o f the vein between the two lengths of suture with a #11 scalpel blade. The appropriate sized catheter was passed in to the vein and secured by lig a tin g the d is ta l suture. The same catheterization procedure was performed on the external ilia c artery in the puppies and the medial circumflex femoral artery in the adult dogs. When all four catheters were in place and secured, the animals were euthanized by administering an intravenous overdose of pentobarbital sodium.

Sixty m illilite r syringes of warm, heparinized 0.9% saline (7,000

USP units/250 ml) were connected to the arterial catheters and the vessels were slowly flushed until clear fluid flowed freely from the venous catheters. In 4 newborn puppies and 2 ad ult dogs, vascular saline flushing was followed immediately by perfusion with a 1% g lu ta r- aldehde-2% paraformaldehyde solution (pH 7.4) to fix the tissues for future scanning electron microscopic (SEM) studies. Approximately 1 ml/kg of body weight of the glutaraldehyde-paraformaldehyde mixture was perfused antegrade through syringes attached to the arterial catheters, followed by retrograde perfusion of the glutaraldehyde-paraformaldehyde mixture (approximately 0.2 ml/kg of body weight) through syringes attached to the venous catheters. M icrofilr , a colored liquid silicone mixture that polymerizes intravascularly after a catalyzing agent is added, was prepared for vascular injection. In general, white

M ic r o filr (MV-112) was used fo r antegrade a rte ria l in je c tio n s , and orange M ic r o filr (MV-117) was used fo r retrograde venous in je c tio n s . 10

The MV compound (pigmented component) was combined w ith MV d ilu e n t and curing agent at a ratio of 4.0:5.0:0.45 vol:vol:vol, and mixed thor­ oughly. Under constant gentle hand pressure, M ic r o filr in je c tio n s were promptly made via antegrade routes (through the external ilia c or med­ ial circumflex femoral arteries), retrograde routes (through the common ilia c or femoral veins), or both antegrade and retrograde routes.

Arterial microcirculation supplying the medullary cavity was to be demonstrated by the antegrade injections; venous microcirculation draining the medullary cavity was to be demonstrated by the retrograde in je c tio n s . The volume o f M ic r o filr used fo r each in je c tio n varied greatly, depending on the size of the animal, the route of injection, and the intended specific vascular structures (arterial or venous) or areas (marrow or cortical bone) to be studied. A minimum of 6 hours post-injection at room temperature was allowed for the intravascular catalization process to occur.

The femurs were harvested, the surrounding soft tissues were removed, and the la te ra l or cra nial surfaces were sectioned lo n g i­ tu d in a lly to expose the marrow c a v ity fo r b e tte r penetration o f the

Spalteholz clearing solutions which were to follow. Multiple cross- sections of the mid-diaphysis were also made on one pair of glutaral- dehyde-fixed adult femurs. A modified Spalteholz clearing technique4>

5 was used to render transparent (cle a r) the osseous and s o ft tissues which permitted three-dimensional visualization of the opaque,

M icrofilr-fille d vascular structures. All specimens except the glutaraldehyde-fixed newborn femurs were thoroughly d e c a lc ifie d in 3% nitric acid. 11

The time required for complete decalcification to occur varied from several hours in the newborn femurs to over one week in the femurs of ad ult dogs. N itr ic acid solu tions were changed d a ily . F atty marrow was removed by submerging the specimens in T rito n X-100 (SEM specimens) or chloroform. The tissues were dehydrated by daily serial passage through successively increasing concentrations of alcohol (50%, 75%,

85%, 95%, absolute). The specimens were cleared and stored in water- fre e methyl s a lic y la te . For microscopic examination, specimens were transferred to an optically correct glass viewing box containing methyl salicylate and illuminated by two fiber optics light sources. Photo­ graphs were taken through a stereoscopic dissecting microscope and an operating microscope using 35mm Zeiss cameras and ASA 50 tungsten color slide film . Scanning electron microscopy was performed on glutar- aldehyde-fixed vessels in nondecalcified fragments of the newborn puppy femurs that had been previously defatted (Triton X-100) and dehydrated

(Spalteholz alcohol sequence). Decalcified, defatted, and dehydrated pieces o f femoral bone from two a d u lt dogs were also prepared fo r scanning ele ctron microscopic examination. The SEM specimens were critica lly point dried, mounted, sputter-coated with gold, evaluated under the electron microscope, and photographed with Polaroid type 55 positive/negative film . 12

RESULTS

The femurs o f the newborn puppies measured approximately 3 mm in diameter at the mid-diaphysis, and 22 mm in length. Both epiphyses were e n tire ly c a rtila g in o u s . Between the two epiphyses, a marrow cav­ ity was present within a shell of periosteal woven bone. The proximal femoral physis was continuous between the femoral head and the greater trochanter. As the puppies became older, the configuration of the femurs changed by disproportional lengthening o f the shaft and non- symmetrical growth at the proximal physis (Fig. 1.1). The increased rate of growth of the proximal physis at the femoral head caused elon­ gation of the femoral neck and the eventual division (at two weeks of age) o f the proximal physis in to separate physes fo r the femoral head and the greater trochanter. The femurs of the adult dogs measured

12-14 mm in diameter (at the narrowest point on the diaphysis) and

165-220 mm in length. From birth to maturity, the femurs displayed a

4.6-fold increase in diameter and a 7.5-10-fold increase in length.

There was a 40-50-fold increase in body weight.

Antegrade injections of white M icrofilr into the medial circumflex femoral artery of adult dogs resulted in the fillin g of arterial ves­ sels and sinusoids w ith in the marrow c a v ity (F ig. 1 .2 ). Attempts at direct cannulation of the diaphyseal nutrient artery of the femur were abandoned due to the d iffic u lt location of the nutrient foramen on the caudal surface of the femur under the attachment of the adductor magnus 13

Figure 1.1

Comparative sizes and configurations o f the femur in the newborn puppy (A), two-week-old puppy (B ), ten-week-old puppy (C ), and adult dog (D) FIGURE 1.1 Figure 1.2

Caudal one-half, proximal le ft femur

(cut surface); adult dog. Low magni­

fication of the arterial vascular

pattern within the medullary cavity.

Arterial structures and sinusoids

are filled with M icrofilr. 16

FIGURE 1.2 et brevis muscle. Upon entering the marrow cavity, the diaphyseal

nutrient artery divided promptly into ascending and descending medul­ la ry a rte rie s . These tortuous main branches soon gave ris e to several smaller, long, longitudinally directed arteries which traveled proxi­ mal ly and distally through the marrow toward the cancellous bone of the proximal and d is ta l metaphyses (F ig. 1 .3 ). Even though most venous structures were not fille d by this injection route, a large concentra­ tion of arterial vessels and sinusoids were present in the adult marrow, making i t extremely d if f ic u lt to c le a rly id e n tify the te r­ minations of the arterial microcirculation. After following the m aturation o f the s im p lifie d a rte ria l and venous marrow systems from birth, a retrospective examination of the marrow vessels in the adult dog was more enlightening.

By only p a r tia lly f i l l i n g a rte ria l and venous marrow systems in the newborn puppy, several antegrade and retrograde injections revealed the generalized vascular patterns within the marrow cavity. The dia­ physeal nutrient artery usually was single, entering the marrow cavity from the caudal surface of the proximal femur and branching into ascending and descending medullary arteries. When proximal and distal diaphyseal arteries were present (4 of 24 puppies), the proximal dia­ physeal nutrient artery gave rise to the ascending medullary artery and the distal diaphyseal nutrient artery gave rise to the descending medullary artery. In the newborn puppy, thin branches arose from the medullary arteries at right angles.

The basic venous drainage system of the medullary cavity within the femoral shaft consisted of a central medullary vein which was Figure 1.3

Caudal one-half, diaphysis of le ft femur (cut surface); adult dog.

Low magnification of longitudinal branches arising from the descending medullary arteries and passing dis- tally within the medullary cavity.

Note where the n u trie n t a rte ry enters the medullary cavity. FIGURE 1.3 20 consistently drained through the cortex by proximal and distal emissary metaphyseal veins. The central medullary vein was also inconsistently drained by very small proximal diaphyseal emissary veins (all passing through the nutrient foramen) and a larger, single distal emissary diaphyseal vein (Fig. 1.4). By selective arterial and venous injec­ tions, close inspection of the nutrient canal in the femurs of adult dogs confirmed the absence ( in most cases) of a large diaphyseal "nu­ trie nt" vein passing through the nutrient canal beside the diaphyseal

nutrient artery. Nearly the entire diameter of the nutrient canal was occupied by the diaphyseal nutrient artery (Fig. 1.5). Several very flattened, narrow veins were demonstrated, however, in intimate contact w ith the diaphyseal n u trie n t a rte ry as i t passed through the n u trie n t canal. Occasionally wrapping around the diaphyseal nutrient artery were fla tte n e d venous plexuses th a t communicated w ith the narrow veins within the nutrient canal (Fig. 1.6).

As the newborn puppies matured, regional drainage patterns became better established in the proximal and distal metaphyses. The marrow of the proximal metaphysis was mainly drained by the large cranial proximal metaphyseal emissary vein (on the cranial surface of the proximal femur) and the large caudal proximal metaphyseal emissary vein, which emerged from the medullary cavity out of the trochanteric fossa. The marrow o f the d is ta l metaphysis was re g io n a lly drained by one to three distal metaphyseal emissary veins, all emerging from the cortex on the caudal surface of the femur (Figs. 1.7, 1.8). The venous anastomoses o f the proximal and d is ta l emissary metaphyseal veins (and the proximal and distal diaphyseal emissary veins, when present) with Figure 1.4

Medial surface view, diaphysis of the le f t femur; newborn puppy. Only the main a rte ria l and venous stru c­ tures of the medullary cavity have been injected. CMV = central medul­ lary vein, DMA = descending medul­ lary artery, PDV = proximal diaphy­ seal emissary vein, DDV = distal diaphyseal emissary vein. FIGURE Figure 1.5

Medullary cavity and lateral cortex

(C) a t the leve l o f the n u trie n t canal; adult dog. The nutrient artery occupies nearly the entire nutrient canal.

Figure 1.6

Longitudinal section through the cortex (C) near the nutrient canal

(NC); adult dog. Several small proximal diaphyseal emissary veins (PDV) surround the transparent nutrient a rte ry . FIGURE 1.5

FIGURE 1.6 Figure 1.7

Craniolateral edge of (laterally sectioned) proximal le ft femur; two-week-old puppy. Cranial (crPMV) and caudal (caPMV) proximal meta­ physeal emissary veins draining the proximal marrow c a v ity (M) and anastomosing with the central medullary vein.

Figure 1.8

Caudolateral edge of (laterally sectioned) distal right femur; two- week-old puppy. Distal metaphyseal emissary vein (DMV) passing obtusely trough the cortex (C) and draining the distal medullary cavity FIGURE 1.7 FIGURE 27 the central medullary vein were retained throughout the development of the femur to serve as permanent collateral drainage routes from the marrow cavity in the femurs of the adult dogs (Fig. 1.9).

The level at which marrow blood flowed from the arterial micro­ c irc u la tio n in to the venous drainage system was best seen in the metaphyses of partially injected newborn puppy specimens. Because a large percentage of vascular structures were not fille d in a few of these specimens, it was possible to trace individual vascular pathways continuously from the a r te ria l system to the venous system in the areas of hematopoietic marrow. Increasingly narrower branches of the medul­ lary arteries, destined to supply the hematopoietic marrow, ultimately terminated in very small arterioles or capillaries which supplied dilated, irregular vascular structures, the sinusoids. Developing sinusoids in the metaphyses emptied into collecting sinuses which in turn converged and le ft the marrow cavity as metaphyseal emissary veins

(Figs. 1.10, 1.11).

Ultrastructural examination of vascular structures within bone marrow revealed c h a ra c te ris tic diffe ren ces between a rte ria l and venous vessels. Arteries were smooth, nearly parallel-walled vessels of gradually changing caliber with numerous longitudinally oriented endo­ thelial nuclei protruding into their lumen. Venous structures, parti­ cularly the sinusoids, exhibited irregular walls and abrupt variations in diameter (Fig. 1.12). Sinusoids appeared to develop by budding dilatations off of collecting sinuses and were very closely associated with the hematopoietic marrow in the interstices of cancellous bone

(Figs. 1.13, 1.14). In the adult specimens, the narrow arteriolar 28

Figure 1.9

Drawing o f the emissary venous drainage of the central medullary vein; adult dog

A = caudal proximal metaphyseal emissary vein

B = cranial proximal metaphyseal emissary vein

C = diaphyseal emissary vein accompanying diaphyseal nut­ rient artery

D = distal metaphyseal emissary vein

E = distal epiphyseal emissary vei n FIGURE 1.9 Figure 1.10

Lateral (cut) surface of distal metaphysis, right femur; newborn puppy. Fine branches from descendi medullary arteries (DMA) supplying sinusoids (VS) just proximal to the distal physis.

Figure 1.11

Lateral (cut) surface of distal metaphysis, le ft femur; newborn puppy. Distal metaphyseal sinu­ soids (VS) converging on c o lle c tin g sinuses) and draining through the caudal cortex via a distal metaphyseal emissary vein (DMV). FIGURE 1.10 FIGURE 1.11 Figure 1.12

SEM o f marrow c e lls and tra becu lar bone; newborn puppy. Comparison of a large smooth-walled artery

(AR) and an irregular sinusoid 33

FIGURE 1.12 34

Figure 1.13

SEM o f tra becu lar bone (TB) and marrow tis s u e ; newborn puppy.

Developing sinusoids (VS) and collecting sinuses.

Figure 1.14

SEM of decalcified trabecular bone

(TB), marrow tis s u e , sinusoids (VS), and collecting sinuses (CS); adult dog. Several collecting sinuses converge on large collecting vein (CV). FIGURE 1.13

FIGURE 1.14 36 capillaries supplying the sinusoids often appeared quite long and

sometimes arose from s u rp ris in g ly la rg e r a rte rie s at very acute angles

(Figs. 1.15, 1.16). Frequently several sinusoids were supplied by a

single capillary and more than one capillary would supply a single

sinusoid. The relatively long arteriolar capillaries supplying sinusoids in the marrow of adult dogs appeared to be extensions of much shorter arteriolar carpillary side branches from arteries in the marrow o f newborn puppies, as seen under the scanning ele ctro n microscope.

(By removing the arterial wall, numerous endothelial nuclear

impressions in the M icrofilr identify this vessel as an artery.)

(Figs. 1.17, 1.18.)

At high ultrastructural magnification, the outer surfaces of

sinuoids and collecting sinuses were wrinkled and featured migration pores fo r the transmural passage o f marrow c e lls from the hematopoietic marrow compartment in to the blood stream. M igration pores were c ra te r­ like in appearance, exhibiting a variable degree of elevation of the

sinusoidal wall around each pore (Figs. 1.19, 1.20). Bi-concave eryth­ rocytes and leukocytes were randomly fixed to the sinusoidal wall. Mi­ gra tio n pores beneath the attached marrow blood c e lls were v is ib le in various stages of development. A large surface area of individual marrow blood c e lls appeared to make in it ia l contact w ith , and become fixed to the sinusoidal wall. As cavitation of a migration pore be­

neath an attached cell became greater, the attached cell apparently began to separate from the sinusoidal wall. Concurrently, the sinus­ oidal wall appeared to be forming a smooth elevation around the peri­ phery of the developing migration pore. When a single stalk was all Figure 1.15

Arterial vessels of the distal

diaphyseal marrow; adult dog.

Long side branches of a large medullary artery (DMA) continu­

ing to divide into narrow arter­

iolar capillaries to supply well-

defined sinusoids.

Figure 1.16

Arterial supply to the sinusoids

(VS) through very narrow capillaries

(cap); adult dog. Ar = arteriole. FIGURE 1.15

FIGURE 1.16 Figure 1.17

SEM o f bone marrow; newborn puppy.

Large artery (AR) lying adjacent to trabecular bone (and near a col­ lecting vein, CV) and giving off acute, narrow arterioles (ar) to supply sinusoids (VS). CS = collecting sinus

Figure 1.18

SEM o f bone marrow; newborn puppy.

Endothelial nuclear protrusions into the lumen of the artery have created impressions in the M icrofilr material. VS = sinusoid, ar = a rte rio le 40

FIGURE 1.17

FIGURE 1.18 Figure 1.19

SEM of a branched collecting sinus (CS) w ith in marrow tis s u e ; newborn puppy*

Figure 1.20

SEM of migration pores (mp) within the walls of a collecting sinus;

newborn puppy. FIGURE 1.19

FIGURE 1.20 43 th a t remained o f the attachment between the marrow blood c e ll and the elevated rim of the migration pore, the marrow blood cell began its transmural migration. Erythrocytes entering migration pores became s lig h tly wedge-shaped by developing an elongation a t th e ir leading edge

(Figs. 1.21., 1.22).

In addition to the arterial capillary-venous sinusoid communica­ tion found throughout the entire marrow cavity (but more heavily con­ centrated in the hematopoietic marrow o f the metaphyses and around the endosteal surfaces), a second type o f a rte ria l venous communication was present. Patches of anastomotic capillary-venule beds were also iden­ t if ie d throughout the marrow c a v ity o f adult dogs, and were b e tte r developed in the mid-diaphyseal zone of fatty marrow. Very few ca p illa ry -v e n u le beds were found in the puppy femur specimens because essentially all of the marrow cavity was fille d with hematopoietic marrow, i. e . , there were no zones o f fa tty marrow. The c a p illa ry - venule beds were composed o f irre g u la r hexagonal u n its th a t were sup­ plied by delicate capillaries arising from the terminal branches of medullary arterioles. Venules draining the capillary-venule beds emptied into larger collecting veins and several collecting veins joined to empty into the central medullary vein (Figs. 1.23, 1.24).

(Note that the term "collecting veins" is used instead of "collecting sinuses." The venous structures draining the capillary-venule beds were designated as "collecting veins" because they lacked the ultra- structural features of collecting sinuses and sinusoids.) Several small patches o f c a p illa ry-ve n u le beds were found between large clumps of hematopoietic marrow in the newborn puppy specimens. Figure 1.21

SEM of a sinusoidal wall; newborn puppy. Erythrocytes (RBC) adhering to sinusoidal walls and passing through migration pores (mp).

Figure 1.22

SEM of the transmural migration o f marrow erythrocytes (specimen has been ro ta te d ). Newborn puppy. FIGURE 1.21

FIGURE 1.22 46

Figure 1.23

Vascular pattern of fatty marrow, mid-diaphysis; adult dog. Numerous venules drain patchy capillary beds.

C = cortex

Figure 1.24

C a p illa ry beds (cb) o f fa tty marrow; adult dog. Several collecting veins

(CV) receive venules (ven) from capillary beds. FIGURE 1.23

FIGURE 1-24 48

Ultrastructural examination further demonstrated the three-dimensional

anastomotic arrangement of the vascular beds and the larger collecting

veins. Venules of the capillary-venule beds lacked migration pores and

exhibited a relatively smooth outer surface as compared to the sinus­ oids and collecting sinuses (Figs. 1.25, 1.26). s' Structural interrelationships of the venous microcirculation with­

in the marrow c a v ity were demonstrated by the retrograde venous in je c ­

tion route. Many sinusoids in the diaphyseal marrow cavity drained directly into the central medullary vein through thin, straight draining venules rather than the typically dilated, irregular meta­ physeal collecting sinuses. Single sinusoids or small groups of sinus­ oids were drained by individual (often very long) drainage venules

(Fig. 1.27). Nearby sinusoids were joined together by intersinusoidal communication vessels th a t were very s im ila r in appearance to the sinusoid-central medullary vein drainage venules. These thin

intersinusoidal communications appeared to frequently join sinusoids located around the periphery of the marrow cavity (endosteal surface)

(F ig. 1.28). By electron microscopy, the marrow o f newborn puppies displayed long and short intersinusoidal communicating venules passing between developing sinusoids (F igs. 1.29, 1.3 0). Communicating venules joined large dilated sinusoids with stringy masses of tortuous venules which drained patchy capillary-venule beds (Figs. 1.31). Occasionally, adjacent capillary-venule beds were also joined by individual communicating venules (Figs. 1.32, 1.33).

As the newborn puppy's microcirculation matured, significant changes occurred in the vasculature o f the proximal and d is ta l Figure 1.25

SEM of a small capillary-venule bed in the marrow c a v ity ; newborn puppy. CV = collecting vein.

Figure 1.26

SEM of anastomotic, three-dimensional venule p a tte rn ; newborn puppy, ven = venule. 50

FIGURE 1. 25

FIGURE 1.26 Figure 1.27

Sinusoids (VS) draining directly into the central medullary vein (CMV) via long and short drainage venules; two-week-old puppy. C = cortex.

Figure 1.28

Transcortical view of intersinu­ soidal communicating venules (com) between several sinusoids (VS).

Two-week-old puppy. ..likiM

FIGURE 1.27

FIGURE 1.28 Figure 1.29

SEM o f trabecular bone; newborn puppy. A long, thin intersinu­ soidal communicating venule (com) joining two sinusoids (VS). Devel­ oping sinusoids draining into a collecting sinus (CS).

Figure 1.30

SEM of collecting sinus (CS) receiving a branched sinusoid

(VS); newborn puppy. TB = trabecular bone. FIGURE 1. 29

FIGURE 1.30 Figure 1.31

Venous structures w ith in the marrow cavity; adult dog. Several capil­ lary-venule beds (cvb) entering collecting veins (CV) beneath the cortex at the level of the nutrient canal. VS = sinusoid, C = cortex. FIGURE 1.31 Figure 1.32

A large tuft of venules joined to a smaller venule tu ft by a single communicating venule (com); adult dog. cvb = ca p illa ry-ve n u le bed,

CV = collecting vein

Figure 1.33

A single dimpled sinusoid (VS) with venules leading from the sinusoid to the central medullary vein (CMV) and a capillary-venule bed (cvb); adult dog. FIGURE 1.32

FIGURE 1.33 59 epiphyses. At birth, both epiphyses were composed entirely of hyaline cartilage. Vascular loops consisting of an arteriole, a venule, and a small capillary anastomosis, entered the cartilaginous femoral head and greater trochanter from the peripheral margins of epiphyseal cartilage. The epiphyseal vessels of the femoral head were continuations of subsynovial arteries and veins which trav­ eled along the surface of the developing femoral neck (Figs. 1.34,

1.35). Epiphyseal vessels which entered the cartilaginous distal fem­ oral epiphysis were continuations of perichondral arteries and veins.

The perichondral vessels passed over the lateral and medial surfaces of the femoral condyle and penetrated the hyaline cartilage at the troch­ lear ridge (Fig. 1.36). Vascular loops within the proximal and distal epiphyses were randomly distributed throughout the hyaline cartilage, and gave o ff a few coarse branches. At times, single vessels extended from the ends o f the c a p illa ry loops and crossed the immature physis to anastomose w ith sinusoids w ith in the marrow o f the metaphysis (Figs.

1.37, 1,38). A scanning electron micrograph of the proximal femur of the newborn puppy demonstrated a large vessel traversing the physis, thereby establishing vascular communication between the developing femoral neck and the cartilaginous femoral head (Fig. 1.39). Within two weeks, branches from the randomly distributed epiphyseal vessels had begun to concentrate centrally to supply the rapidly establishing secondary centers of ossification. A majority of the blood volume of the secondary ossification center was contained within venous

structures--venules, veins and a few early sinusoids. Venous drainage of the femoral head was facilitated by a significant increase in the Figure 1.34

Craniolateral surface, le ft fem­

oral head and neck; newborn puppy.

Vessels on the surface of the femor­ al neck penetrating the cartilaginous

femoral head to form random epiphy­

seal vascular loops (EP).

Figure 1.35

Branching vascular loops (EP) with­

in the c a rtila g in o u s femoral head; newborn puppy. Synovial vascular network (SV) on the surface of the femoral neck. FIGURE 1.34

FIGURE 1-35 Figure 1.36

Epiphyseal vascular loops (EP) of the cartilaginous femoral condyle, cranial surface; newborn puppy. Perichondrial ves­ sels (PC) from the lateral and medial surfaces at the trochelar margin penetrating the distal epiphyseal c a rtila g e . FIGURE 1.36 Figure 1.37

Lateral (cut) surface, distal fem­ oral epiphysis; newborn puppy. Ran­ dom distribution of vascular loops within the cartilaginous femoral head.

Figure 1.38

Lateral (cut) surface, distal fem­ oral physis; newborn puppy. Vessels from the ends o f the epiphyseal vas­ cu la r loops (EP) tra versin g the physis and communicating w ith d is ta l meta­ physeal sinusoids (VS). 65

FIGURE 1.37

FIGURE 1.38 Figure 1.39

SEM of the proximal physis; new­ born puppy. A large vein coursing

from the proximal metaphysis, across

the physis (PH) and through the

proximal epiphysis (EP). f ig u r e Figure 1.40

Cranial surface, right femoral head and neck; two-week-old puppy.

Vascular supply to , and venous drainage from, the secondary center of ossification (SC), ac = articular cartilage.

Figure 1.41

Subsynovial veins (DV) on the cranial surface of the femoral neck descending from the secondary center of ossification (SC); two- week-old puppy. FIGURE 1.40

FIGURE 1.41 70 number and diameter of subsynovial veins descending the femoral neck

(Figs. 1.40, 1.41). As the puppies grew older, the secondary ossification centers enlarged. More and more of the random vascular loops within the remaining epiphyseal cartilage were incorporated into the microvasculature of the expanding epiphyseal marrow cavities.

At ten weeks of age, proximal and distal physes were clearly defined and no vascular stru cture s were seen crossing them. Small a rte rio le s and c a p illa rie s advanced to the faces of the physes, only to turn back by joining sinusoids (usually) which drained away from the physes into metaphyseal or epiphyseal collecting sinuses. Vascular loops within the narrowing band of hyaline (articular) cartilage were s till present in the ten-week-old specimens. Openings in the plate of subchondral bone allowed m ic ro c irc u la tio n to occur between the a r tic u la r c a rtila g e and the epiphyseal marrow cavity (Figs. 1.42, 1.43). No vessels re­ mained in the relatively thin rim of articular cartilage in the femoral head and condyle of the adult dogs. It was d ifficu lt to identify the location of the former proximal physis due to the continuity of vas­ cular structures between the femoral head and neck. In general, the marrow c a v ity of the femoral head had an even d is trib u tio n o f hemato­ poietic and fatty marrow vascular arrangements. Continuity of the microcirculation was best seen in the subchondral bone where arterial capillaries drained into sinusoids or venules (Figs. 1.44). At no time during the development of the femur was the epiphyseal vascular supply augmented by penetration of the articular cartilage from vessels within the ligament to the head of the femur. On the contrary, all of the numerous arterial vessels in the ligament formed blind ending loops with venous structures at the fovea of the femoral head (Fig. 1.45). 71

Figure 1.42

Lateral (cut) surface of distal

femur; ten-week-old puppy. Devel­

oping secondary ossification center

(SC) incorporating more epiphyseal cartilage and giving better defini­ tion to the physis (PH) and articu­

lar cartilage.

Figure 1.43

Vessels of the secondary ossification center; ten-week-old puppy. Residual

vascular loops within the epiphyseal cartilage (EC) are continuous with the vessels of the expanding secondary ossification center across a plate of

subchondral bone (SB). EA = distal epiphyseal nutrient artery, EV = distal epiphyseal emissary vein. FIGURE 1.42 FIGURE 1.43 73

Figure 1.44

Caudal articular surface, femoral head; adult dog. Subchondral vascu­ la r c o n tin u ity between a rte ria l cap­ illa rie s (cap), sinusoids (VS), and collecting sinuses (CS).

Figure 1.45

Vascular loops within the ligament to the head of the femur (LIG); adult dog. Ligament vessels are blocked from epiphyseal vessels

(EP) by a plate of articular cart­ ilage (ac). 74

FIGURE 1.44

FIGURE 1.45 Figure 1.46

Drawing o f the a rte ria l and venous marrow m ic ro c irc u la tio n ; newborn puppy.

A = cranial proximal metaphyseal emissary vei n

B = diaphyseal nutrient artery

C = central medullary vein

D = collecting sinus

E = distal metaphyseal emissary vein

F = arteriolar capillaries

6 = distal epiphyseal vascular loops 76

FIGURE 1.46 Figure 1.47

Drawing of microvascular relation­ ships at the proximal and distal physes; ten-week-old puppy.

Proximal femur:

A = vascular loops in the remaining epiphyseal cartilage

B = sinusoids and collecting sinuses in the secondary ossification center

C = superficial periosteum, reflected

D = cranial proximal metaphyseal emis­ sary vein

E = diaphyseal nutrient artery

Distal femur:

A = central medullary vein

B = distal metaphyseal emissary veins

C = distal epiphyseal emissary vein

D = distal physis FIGURE I. 4 7 79

DISCUSSION

Terminology. D iffe re n t in ve stig a to rs have used various terms when referring to the vascular structures within the medullary cavity. This author chose to use descriptive terms which identified the type of vessel (arterial, venous), suggested its approximate size and character, and expressed its general location relative to the ends or shaft of a long bone.

The term "nutrient artery" commonly refers to the principal artery entering the medullary cavity. All arteries that pass through nutrient foramina are "n u trie n t" vessels, i. e . , they nourish osseous and marrow tissues. In an attempt to be more specific, arterial vessels entering the medullary cavity at different levels were referred to as "epiphy­ seal," "metaphyseal," or "diaphyseal nutrient arteries." The common term "nutrient vein" is contradictory.6 "Epiphyseal," "metaphyseal," or "diaphyseal emissary veins" (referring to veins draining the medul­ lary cavity by passing directly through the cortex) was preferred.

Rather than the usual single diaphyseal nutrient artery of the femur in the dog,^ in this investigation 4 of 24 puppies demonstrated dual dia­ physeal nutrient arteries of nearly equal size located proximally and distally on the caudal aspect of the femoral diaphysis. Also, all long bones have an epiphysis, a physis (in growing bones) and a metaphysis at their proximal and distal ends. Therefore, the use of "proximal" and "distal" to further describe the location of vessels in these areas was considered appropriate.

Very narrow branches from medullary arterioles which supplied si­ nusoids have been previously referred to as "thin-walled arteries."6*® As shown with transmission electron microscopy, the walls of "thin- walled arteries" are usually only two cell layers thick, an endothelial lining and a single layer of smooth muscle cells.9 In this investiga­ tion these vessels were considered to be arteriolar capillaries, be­ cause the tunics (intima, media, adventitia) of typical arteries are not present. Vessels of similar size and configuration to capillaries o f s o ft tissues elsewhere were c h a ra c te ris tic a lly seen in fa tty marrow areas and as side branches from radiating intracortical arterioles (see

Chapter II, Results).

Narrow, straight vessels joining adjacent sinusoids or individual sinusoids to the central medullary vein were interpreted as small venous stru ctu re s. Previous in ve stig a to rs T0*1! have re ferred to the intersinusoidal vascular communications as "capillaries"—an unlikely s itu a tio n , since the vessels are interposed between two venous stru c­ tures. This author considered intersinusoidal "communicating venules" a more accurate designation. Vessels of sim ilar morphology which drained blood from individual sinusoids directly into the central medullary venous system were ca lle d "drainage venules." The term

"collecting sinuses" was reserved for irregular vascular structures which drained sinusoids and exhibited migration pores in their wrinkled, thin walls. Collecting sinuses had some features in common with sinusoids, but were not sufficiently dilated nor blind ending to justify the term "collecting sinusoids." "Collecting veins" differed from collecting sinuses by demonstrating no migration pores in their smoother, less irregular vascular walls. Blood from venule beds (fatty marrow) drained into collecting veins. Because transmural migration also apparently does not occur in the principal venous structure within the marrow c a v ity , the use o f "central medullary vein" ra th e r than

"central medullary sinus" or "central medullary sinusoid"12 was preferred.

Vascular Structure and Function. Most investigators now believe that tissues within the medullary cavity are exclusively supplied by epiphyseal, metaphyseal, and (particularly) diaphyseal nutrient arteries.13*14 Others have reported that marrow nutrition is augmented to a variable degree by periosteal vessels.9*15»16*1^ Although vascu­ la r stru cture s were shown to jo in the periosteal and marrow vascular systems (see Chapters II and III), evidence for centripetal transcorti- cal blood flow from the periosteum to marrow tissue was not found. In newborn puppies only, arterial supply to the medullary cavity was mini­ mally supplemented by perichondrial vessels at the synovial margins of the joints. The vessels penetrated proximal and distal epiphyseal car­ tila g e s as random vascular loops. Small c a p illa rie s from the ends o f the vascular loops crossed the physis and anastomosed w ith metaphyseal sinusoids. The transphyseal vascular connections were transient, and could not be demonstrated in specimens two weeks of age and o ld e r.

While apparently not a common clinical problem in newborn puppies, transphyseal vascular anastomoses represent a p o te n tia l route o f bone marrow infection from an infectious synovitis, as seen in the septic arthritis-osteom yelitis syndrome of neonate foals and children.18*19

Arteriolar capillaries leading to sinusoids were unique in their narrowness (as compared to their parent arteries), acute angle of o ri­ gin, and unusually long length. The abrupt decrease in arteriolar capillary wall thickness has suggested significant vessel distensi- b ility during systole, causing a pulsatile increase and decrease in adjacent marrow tissue f lu id pressure. The a lte rn a tio n s in marrow tissue fluid pressure are thought to aid in the transmural migration of marrow blood cells.20 Another possible function of the arteriolar capillaries is suggested by their narrowness, long length, and acute angle of origin. The hematocrit of intrasinusoidal blood is apparently lower than the hematocrit of extraosseous blood.2! , 22,a hypothetical explanation for this discrepancy involves the concept of "plasma skimming" by the arteriolar capillaries.23 Plasma is "skimmed off" of blood flowing through the large diameter parent arteries and directed through the arteriolar capillaries into the sinusoids. The right-angle origin of arteriolar capillaries may increase the efficiency of the skimming process.9 During a strong erythropoietic or granulopoietic stimulus, "showers" of cells may be released into the hypocellular intrasinusoidal blood, causing an increase in the hematocrit of the blood leaving the sinusoids.2^

Two investigators have described "occasional" arterio-venous shunts from the arteriolar capillaries to post-sinusoidal vessels. The shunts apparently by-pass sinusoids and capillary venule beds.16*25

Morphological evidence for arteriovenous shunting was not found in this investigation. The significance of arteriovenous shunts within the marrow—if they exist—remains speculative at this time.

Sinusoids are extremely thin-walled (one cell layer thick), large diameter vessels that are largely responsible for the preponderance of venous blood w ith in the hematopoietic marrow tis s u e .8*9 The 83

cross-sectional ratio of a rte ria l:venous structures has been estimated

to be 1:30 in the long bones of guinea pigs,26 but the ratio of arte­

riolar: sinusoidal blood flow is 20:1.27 Undoubtedly, the site of entry

fo r fa t and marrow emboli a fte r a long bone fra c tu re , or to ta l hip re­ placement, is through tears in the thin-walled, large diameter sinus­ oids. 28,29,30 7 he radiographic technique of intraosseous venography developed by Steinback and others31>32 -js possible because the major p o rtio n o f intram edullary blood volume is contained w ith in venous structures. Radiographic contrast material injected through the cortex

into hematopoietic marrow areas results in fillin g of the sinusoids and, eventually, the remainder of the marrow venous system. Intersi­

nusoidal communicating venules probably help distribute the contrast material throughout the medullary cavity.

Sinusoids appear to be vascular extensions of collecting sinuses because both have a very similar wall structure. Both types of vessels have simple endothelial linings which are capable of forming transient migration pores for transmural cellular migration.33»3^»33 The endo­ thelial cells of sinuoids and collecting sinuses are also capable of phagocytosis.36

Marrow c o lle c tin g veins and the central medullary vein e xh ib ite d a more structured vascular wall. These veins were s till quite thin- walled and distensible when compared to their arterial counterparts.11

Because marrow veins are re a d ily d is te n s ib le , changes in systemic ven­ ous pressure are ra p id ly transm itted to the marrow tis s u e . Marrow venous pressure is therefore e s s e n tia lly the same as marrow tissu e

fluid pressure.37 84

Vascular S tructure and Marrow Blood Flow. In ve stig a tin g the dyna­ mics o f marrow blood flow by in vivo methods is te c h n ic a lly d if f ic u lt .

Branemark and K inosito were able to observe blood flow ing through the marrow sinusoids in the long bones of rabbits.27*38 Sinusoidal blood flow was characteristically irregular, with alternating periods of sluggish flow and stagnation. Sinusoids rhythmically dilate over a period of 1-2 minutes, followed by a slightly faster emptying period.

Flow rates through the sinusoids were 0-0.2 mm/sec, as compared to arteriolar flow of 1.0-1.5 mm/sec and capillary flow of 0.5 mm/ sec.27

With an overrepresentation of sinusoids in the metaphyseal regions of a long bone, a p re d ile c tio n fo r (hematogenous, se p tic ) osteom yelitis and metastatic bone tumors at these sites is predictable.39*40 Bacteria or neoplastic cells could better establish themselves in areas of stagnate blood flow. Contact time would be prolonged and the circulating body defense mechanisms (a ntibo dies, complement system fa c to rs , leukocytes, macrophages) would be decreased in these areas.

As described in Chapter I , Results, the basic venous marrow drainage system from emissary veins is well established at birth (Fig.

1.46). The volume of blood that can be drained by any individual emis­ sary vein is severely limited by the incapacity of the vein to appreci­ ably dilate within the rigid confines of the emissary canal. Marrow veins do not possess valves, so that blood entering the central medul­ lary vein may be shunted proximally or distally, depending on regional pressure gradients. Muscular activity is suspected to increase extra- osseous vascular resistance, particularly along the diaphysis.4! If vascular resistance is increased along the shaft of a long bone, 85

increased venous outflow from metaphyseal emissary ve in s --w ith in lim -

its --c o u ld prevent vascular engorgement o f the marrow m icro circu la ­ tio n . In the a d u lt, venous blood in the central medullary vein may also be drained by epiphyseal emissary veins due to the vascular c o n tin u ity between the metaphyseal and epiphyseal marrow. From less than 2 weeks of age until skeletal maturity (during active endochondral bone formation) arterial and venous systems of the epiphysis and metaphysis are segregated above and below the physis (Fig. 1.47).

Marrow blood flow varies greatly in different locations within the medullary cavity. The variation is grossly related to the fatty or he­ matopoietic character of the marrow tissue. Blood flow is much greater in hematopoietic marrow than in fatty marrow.42 Since the medullary cavity of puppies is composed almost entirely of hematopoietic marrow, and hematopoietic marrow progressively changes to fa tty marrow during the aging process, marrow blood flow is also an inverse function of age. Neural, humoral and metabolic fa cto rs also have an e ffe c t on regional blood flow.43 Vascular resistance has been shown to increase with exercise, hemorrhage, norepinephrine and sympathetic stimula­ tion .44 Increased marrow blood flow may occur with increased vascular resistance when the lim its of venous drainage from other emissary veins have been exceeded. Recent exercise experiments in conscious dogs have shown a 65% increase in marrow blood flow only after extended physical exercise. The marrow hyperemia persisted fo r one hour a fte r exercised had ceased.45 Gross showed a sim ilar slow response with prolonged exercise and unaltered perfusion rates during moderate exercise of short duration.44 These studies support the earlier belief 86 that metabolic factors causing an increase in pC 0 2, and a decrease in

PO2 and pH are la rg e ly responsible fo r increased marrow blood flo w .46*47

Blood flow in marrow has been d if f ic u lt to accurately measure due to some of the conditions listed above. Different techniques of undoubtedly varying accuracies have been devised to investigate this subject.48 The most accurate technique to date appears to be the use of radio-labeled microspheres. In one study, blood flow was shown to be 19.6 ml/100 Gm/min in the femoral head, 50.3 ml/100 Gm/min in the femoral neck, and 29.0 ml/100 Gm/min in cancellous bone of the tib ia .49

Transmural Migration. Early ultrastructural investigations of the sinusoidal wall suggested that the movement of blood cells from the extravascular marrow across the wall was facilitated by a separation of adjacent endothelial cells at contact points. The sinusoidal walls were described as "functionally incomplete" and in a state of "dynamic flu x and readjustm ent."10 Weiss and DeBruyn have since shown th a t transmural migration is intracytoplasmic, i.e ., through endothelial c e lls , ra th e r than between them. M igration pores are u ltra s tru c tu ra lly described as diaphragmed fenestrae which maintain the functional integ­ rity of the sinusoidal wall during transmural cellular passage.42*50

With transmission electron microscopy, the sinusoidal wall has been shown to consist of a complete layer of lining (endothelial) cells, a discontinuous basal lamina, and a discontinuous layer of adventitial cells. No adventitial cells or basal lamina can be found at migration pore locations.84 The mechanism of migration pore formation is un­ known.3 In this investigation, scanning electron micrographs of the sinusoid's marrow surface suggested a surface attraction and attachment of mature marrow blood cells to the sinusoidal wall. Erythrocytes had already shed their nuclei before attaching to the sinusoidal wall, contrary to the observation of Weiss in which nuclear extrusion was said to occur as a result of erythrocytes squeezing through the very narrow migration pores.42 Subsequently, the concurrent formation of a migration pore beneath an attached erythrocyte and the sequential detachment of the erythrocyte appeared to occur. If migration pores do form in response to the attachment of mature marrow blood cells, empty migration pores would represent recent transmural cellular migrations or pores which have lost their marrow blood cells during tissue processi ng.

C ells th a t pass from hematopoietic marrow in to the vascular com­ partment are described as "myelofugal." There is some evidence to sug­ gest that a few intravascular lymphocytes attach to the luminal surface of the sinusoidal wall and pass through the wall into the extravascular marrow ("m yelopetal" passage).3 Because these observations were made from static figures, the actual direction of movement of the

"myelopetal" cells remains questionable.

The sinusoidal wall is apparently freely permeable in either di­ rection to large plasma protein molecules and particulate matter.51*52

Since extravascular protein can be easily removed at the sinusoid level by the venous m ic ro c irc u la tio n , the absence o f lymphatic vessels w ith in bone marrow—while not conclusively proven —appears likely. Freely flow ing marrow tissue f lu id , termed "transparenchymal plasma flo w "23 also decreases the need for a marrow lymphatic system and may aid in the transparenchymal migration of nonmotile marrow blood ce lls.3 CHAPTER I I

MATURATION OF THE MICROCIRCULATION IN CORTICAL BONE

88 ABSTRACT

Arterioles arising from medullary arteries traveled directly to the endosteal cortical surface and entered (individually or in multi­ ples) penetrating canals. The cortical arterioles assumed a gener­ alized radial course toward the periosteal surface, where an anasto­ mosis with the deep periosteal plexus occurred. Venules arising from endosteal sinusoids o f the marrow c a v ity also entered the endosteal cortical surface and followed a similar pattern through the cortex to the deep periosteum.

Within the cortex, arterioles gave off smaller arterioles and capillaries to supply longitudinally oriented osteons. Resorption cavities, developing, and mature osteons were identified by their in- tracortical vascular patterns. (Wavy transcortical vascular patterns o f newborn puppy specimens re fle c te d the irre g u la r organization o f woven bone. Longitudinal branches within the cortex did not arise until secondary bone formation began.) Arterioles supplying resorption cavities doubled back within the cavities as venules at the head of the

“cutter cone".

The deep periosteal plexus received blood from radial cortical arterioles and venules, and emptied into veins of the superficial periosteum.

89 90

INTRODUCTION

For many years, most investigators have agreed that arterial supply to cortical bone is mostly or entirely supplied by arterioles within the marrow cavity.1-6 A small portion of the outer cortex may be supplied by periosteal arterioles, particularly in the areas of heavy fascial attachment. The direction of arterial flow is centrifu­ gal, from the endosteal surface to the periosteum.6-8 The presence and source of venous structures within the cortex, the centrifugal or centripetal direction of venous blood flow, and the reversal of flow under certain circumstances are s till controversial issues.9-13 These questions persist because of the technical difficulties encountered in studying the microcirculation of cortical bone, and the debatable interpretation of static vascular patterns relative to actual blood flow dynamics. Intracortical vessels are very small, and are confined to tiny vascular channels encased in a rigid, opaque matrix. To be visualized, the incarcerated vessels must be adequately fille d with an identifiable injection material, and the surrounding opaque, mineralized tissue removed or rendered transparent by radiography or clearing techniques.

Adequate circulation through bone is necessary for osteogenesis, bone growth, maintenance and the satisfactory repair of fractures and other injuries to bone.1^ The performance of some of these functions has been directly related to local metabolic factors: pH, p02, and pC02-1^“ 1^ Consequently, the a rte ria l or venous nature o f the blood and its direction of flow through the cortex becomes very relevant, both physiologically and clinically. Trueta has stated, "I firmly believed and s till do, that the vessels and the blood they carry are the main factors responsible for the normality as well as for the many pathological conditions affecting bone."18 92

MATERIALS AND METHODS

Twelve newborn puppies, 6 two-week-old puppies, 6 ten-week-old puppies, and 17 adult dogs were heparinized, anesthetized, catheterized, euthanized, and perfused with orange or white M icrofilr .

(For details of the procedures, see Chapter I, Materials and Methods.)

Retrograde venous perfusions through the proximal portion of the femoral vein (adult dogs) or external iliac vein (puppies) were in­ tended to identify venous structures in the cortex arising from the marrow c a v ity . Venous communication between the outer cortex and p e ri­ osteum, if present, would also be fille d by the retrograde flow of

M ic r o filr . Orange pigmented M ic ro filr was used fo r the venous studies.

Antegrade arterial injections were made through the external ilia c artery (puppies) or medial circumflex femoral artery (adult dogs) using white pigmented M icrofilr . Based on a previous study,12 fillin g of arterial structures within the cortex from medullary arterioles was expected to occur before the marrow venous system had f ille d .

After injections were completed, only partial decalcification of some of the specimens was perm itted. Since the e n tire thickness o f bone was not cleared, the superimposition of vessels out of the plane of focus was minimized. Certain aspects of the cortical microcircu­ lation in the partially cleared cortex were more easily depicted photographically by th is method. RESULTS

Vascular supply to cortical bone essentially arose from arterial structures within the marrow cavity which traversed the cortex and eventually communicated w ith a periosteal vascular system. C ertain venous stru cture s w ith in the marrow c a v ity likew ise were continuous with the periosteal vascular system. Microvascular patterns within the cortex changed from the newborn puppy to the a d u lt dog. The vascular changes were a re fle c tio n o f the progressive conversion o f weak, woven

(primary) cortical bone of the newborn puppies' femurs, to much stronger secondary bone, characterized by longitudinally oriented systems of osteons and well represented in the femurs of adult dogs.

In the adult dog, some arterioles arising from arteries within the medullary cavity entered penetrating (Volkmann) canals at the endosteal surface of the cortex. (As previously described in Chapter I, Results, other arterioles arose from the medullary arteries and, by entering sinusoids and capillary beds, were responsible for arterial supply to the marrow microcirculation.) On cross-sectional examination, the cortical arterioles were somewhat radially oriented, particularly in outer and inner cortical zones which corresponded to the outer and inner circumferential lamellae. Within the middle zone of cortex, the radial arterioles gave o ff smaller side branches (smaller arterioles and capillaries) which were oriented longitudinally within the central

(haversian) canals of individual osteons. Because of the longitudinal vascular branching in the middle cortical zone, an abrupt demarcation between osteonal bone and circumferential lamellar bone was easily demonstrated (Figs. 2.1, 2.2). Most of the medullary arterioles entered 94 penetrating canals on the endosteal cortical surface individually.

Very frequently, however, multiple arteriolar branches from a small medullary artery would arise simultaneously near the endosteal surface and travel parallel with each other into the cortex through a single large endosteal foramen. The multiple arterioles would usually travel into the osteonal (middle) zone of the cortex before diverging into individual vascular channels (Fig. 2.3). At the endosteal surface, the multiple arterioles would occasionally become quite tortuous

(glomerulus-like) and then straighten out again just before entering their common endosteal foramen (Fig. 2.4).

Venous structures within the cortex were best demonstrated in the newborn, two-week-old, and ten-week-old puppies by using only the ret­ rograde venous injection route. Intracortical venules fille d by retro­ grade flow from venous stru cture s w ith in the medullary c a v ity and the periosteal venous system. Sinusoids located near the endosteal surface gave rise to venules which also entered penetrating canals. (Venules that join endosteal sinusoids and vascular structures within the deep periosteal plexus have been termed "portal" vesselsJ® The term is helpful in distinguishing transcortical venules arising from endosteal sinusoids from venules arising within the cortex which drain cortical capillaries and arterioles.) In the very young puppies, portal venules were comparatively large diameter vessels that gave o ff regular, very short, angular branches as they passed outward through the woven co rti­ cal bone. Outside the cortex the venules emptied into the periosteal venous system (Figs. 2.5 , 2 .6 ). In the ad ult dogs, endosteal sinusoids retained their transcortical venous communication with the periosteum. Figure 2.1

Cross-section of mid-diaphysis; adult dog. Cortical vessels radia­ ting outwardly from the endosteal surface to the periosteum.

Figure 2.2

Cross-section of cranial cortex, mid- diaphysis; adult dog. Demarcation of outer and inner circumferential lam­ ellae from middle osteonal layer.

C = co rte x, M = marrow c a v ity . 96

FIGURE 2.1

FIGURE 2.2 Figure 2.3

Inner cortex and outer marrow cavity (cut surface); adult dog.

Multiple arterioles entering a single penetrating canal at the endosteal surface. C = cortex.

Figure 2.4

Inner cortex and outer marrow cavity (cut surface); adult dog.

Tortuosity of arteriolar branches at the endosteal surface. C = cortex. FIGURE 2.3

FIGURE 2.4 Figure 2.5

Venous structures within the dia­ physeal cortex; ten-week-old puppy.

Endosteal sinusoids (EVS) giving rise to portal venules which traverse the cortex to the deep periosteum.

C = cortex.

Figure 2.6

Diaphyseal cortex (C), cut surface; ten-week-old puppy. Cortical venules joining the deep periosteal plexus

(DPP). C = cortex. 100

1*>, S*

FIGURE 2 .5

FIGURE 2.6 However, due to secondary bone form ation, bone growth, and c o rtic a l remodeling, portal venules became less distinctive in their morphology, exhibiting a more delicate and smoother branching pattern (Fig. 2.7).

Therefore, when both a r te ria l and venous vascular in je c tio n s were performed, it was frequently d ifficu lt to differentiate intracortical arterioles and capillaries from venules. Single venules arising from

sinusoids or in d ivid u a l marrow a rte rio le s were traced in to and through the cortex by adjusting the focus of the stereoscopic dissecting microscope. (Unfortunately, available photographic methods used in this investigation limited the depth of focus to a very narrow plane.

Much of the three-dimensional quality of the vascular patterns and the course of individual vessels as seen through the stereomicroscope have been lost in the photographs [Fig. 2.8]). Medullary arterioles usually accompanied venules from endosteal sinusoids in to endosteal foramina, but there appeared to be many more arterioles entering the cortex alone.

In the adult dogs, resorption cavities indicative of bone remodel­

ing were identified by characteristic intracortical vascular patterns.

Very large diameter, longitudinally oriented vascular channels were usually located in the middle (osteonal) cortical zone (Fig. 2.9).

Vessels within the cortex entered or le ft the resorption cavity at different levels. Multiple vessels traveled within the resorption cavity to the "cutter cone" apex (Fig. 2.10). Arterioles at the head of the "cutter cone" turned back within the resorption cavity as ven­ ules. Other narrower longitudinal vascular channels containing fewer vessels were regarded as maturing osteons (Fig. 2.11). Individual capillaries occupied very small central canals within mature osteons. 102

Figure 2.7

Cortex and endosteum (cut surface); adult dog. Portal venules from endosteal sinusoids (EVS) giving off longitudinally oriented side branches as they travel outward through the cortex (C).

Figure 2.8

Cortex (C), endosteum, and outer medullary cavity; adult dog. Cort­ ical arterioles and venules communi­ catin g between the deep periosteum

(DP) and the marrow c a v ity . 103

FIGURE 2 .7

FIGURE 2.8 Figure 2.9

Cortex (C), endosteum, and outer medullary cavity (cut surface); adult dog. Evidence of cortical remodeling by vessels within

several resorption cavities (rc). 105

FIGURE 2.9 Figure 2.10

Cortex (cut surface); ad ult dog.

Tuft of vessels at the "cutter zone head (cc) of a resorption cavity.

Figure 2.11

Cortex (cut surface); adult dog.

Arteriole (art) bifurcating at the apex of a resorption cavity and forming two re turn ing venules (ven) 107

FIGURE 2.10

FIGURE 2.11 A deep periosteal plexus of vessels located in the inner (cellu­ lar) layer of the periosteum received the intracortical venules and terminal branches of the radial arterioles. In the very young puppies, the deep periosteal plexus was highly developed, demonstrating a longi­ tudinally oriented, irregularly rectangular system of vessels. Nearly all of the cortical vessels weaving through vascular channels in the woven bone appeared to anastomose w ith the deep periosteal plexus at regular in te rv a ls (F igs. 2.12, 2.13). Older puppy specimens re fle c te d a progressive decrease in the extent and complexity of the deep perios­ teal plexus (Fig. 2.14). In the adult dog, the deep periosteal plexus was irregularly represented over the surface of the femur. Some longitudinally oriented outer cortical vessels appeared to have been incorporated from the deep periosteal plexus into the cortex by appositional (periosteal) bone formation. The overlying superficial

(fibrous) layer of periosteum contained considerably fewer, but larger, vessels which originated from or drained toward the linea aspera of the femur. Vessels in the superficial layer of periosteum were transversely oriented as compared to the longitudinal orientation of the underlying deep periosteal vessels. Vascular connections between the deep periosteal plexus and the superficial vessels occurred periodically. In the superficial periosteal layer, two veins often traveled parallel to a centrally located artery. In the superficial periosteal veins, blood flow was apparently in a cranial to caudal direction. Systemic veins, located parallel to the linea aspera and within the femoral attachment of the adductor muscles, received blood from the superficial periosteal veins (Fig. 2.15). Figure 2.12

Lateral (cut) surface, right femoral diaphysis; newborn puppy. Cortical vessels emanating from mid-diaphyseal focus. M = marrow c a v ity .

Figure 2.13

Periosteal surface (above) and cut cortical surface (below), mid- diaphysis; newborn puppy. C o rtica l vessels draining into the deep periosteal plexus (DPP). C = co rte x. FIGURE 2.12

FIGURE 2.13 Figure 2.14

Periosteal surface, mid-diaphysis; ten-week-old puppy. Superficial periosteal vessels (SP), deep peri­ osteal plexus (DPP), and endosteal vessels.

Figure 2.15

Periosteal surface, mid-diaphysis; adult dog. Superficial periosteal vessels originating from the linea aspera. AM = adductor muscle. 112

FIGURE 2.14

8

FIGURE 2.15 113

DISCUSSION

From b irth to skeletal m a tu rity, the femur specimens showed a 4.6 fold increase in mid-diaphyseal diameter, and a 7.5 fold increase in length. The body weights of the adult dogs were 40 to 50 times greater than the body weights of the newborn puppies. The disproportional increase of body weight to femur diameter and length placed additional mechanical stress on the femurs. Bone failure under this increased load was prevented by the replacement of woven (primary) bone by the osteonal "pillars" in lamellar (secondary) bone. Wavy vascular patterns indicated the haphazard arrangement of woven bone spicules in the newborn puppies' femurs. Even in the newborn puppies, vascular patterns suggested that blood flowed through the cortical arterioles and venules from the endosteal surface to the periosteum. Vascular anastomoses between the numerous c o rtic a l vessels and an extensive deep periosteal plexus were quite regular and frequent (Fig. 2.16). Many of the e a rly vascular pathways through the woven bone were apparently retained as radiating arterioles in secondary bone. During secondary bone formation, the pre-existing radiating arterioles and venules within the woven cortical bone gave rise to small capillaries and venules which supplied developing osteons. The relative concentration of large radiating arterioles and venules decreased as the volume of secondary cortical bone increased. The decrease in concentration of ra d ia tin g in tra c o rtic a l vessels was accompanied by a re la tiv e decrease in the number of cortical-periosteal vascular anastomoses. Based on the observed differences in concentration of cortical vessels and the number of venous connections with the deep periosteal plexus, very Figure 2.16

Drawing o f the a rte ria l and venous microcirculation in woven cortical bone; newborn puppy.

A = superficial periosteal vessels

B = deep periosteal plexus

C = cortical venules arising from endosteal sinusoids

D = cortical arterioles, from med­ ullary arterioles

E = distal epiphyseal cartilage 115

FIGURE 2.16 116 young puppies would be expected to have much greater cortical blood flow rates than ad ult dogs. Indeed, measurements o f c o rtic a l blood flow in immature and adult dogs have been found to be 7.0 ml/100 Gm/ min and 2.5 ml/100 Gm/ min, respectively.20 (Others have provided evidence for the greater concentration of vascular structures in primary bone than in secondary bone, and a shorter maximum distance between osteocytes and vessels in primary bone than in secondary bone.21>22) One study, using the antipyrine washout technique, suggested that 70 per cent of nutrient arterial blood passed into the cortex and 30 per cent into the marrow.20

In general, radially oriented cortical arterioles may be regarded as "conduit" vessels, and longitudinal vessels within central canals of osteons may be regarded as "nourishing" vessels.^ From transmission electron microscopic studies, vessels within central canals ultrastructurally resemble capillaries and are usually singular.

(Interestingly, unmyelinated nerve fibers with a possible vasomotor function are present in the osteons of adult dogs but absent in the osteons of young puppies. Lymphatic vessels are not present in the osteons of adult dogs or puppies.2^) When two vessels are present within the same central canal, one vessel is significantly larger and th in n e r walled than the o th e r.20 Branemark observed blood flow ing in opposite directions when two vessels were contained within the same central canal.20 The developing osteons containing an arteriolar capillary and returning venules identified in this investigation offered compatible anatomic evidence of bi-directional flow within vessels of individual osteons. Osteons are not truly longitudinal in their orientation, but instead, spiral slightly as they pass down the shaft of the bone.

Within the le ft femur, osteons spiral distally in a clockwise direction; in the right femur the spiral is counterclockwise.27 Spiral resorption cavities may represent a structural weakness in cortical bone, i.e ., a point of failure when torsional forces are applied to the femur. Osteons often end b lin d ly and e x h ib it branching patterns to form anastomosing systems. The osteonal branches appear to form from vascular buds which arise from central canal vessels during the course o f bone remodeling. Vascular changes occur during the form ation and development of new osteons. Multiple vessels within a resorption cavity "drop out" as successive lamellae are laid down along the walls of the central canal.25 In this investigation, several vessels were seen in resorption cavities, two or three vessels in maturing osteons, and single capillaries in mature osteons. (By morphometric analysis, the surface area of vessels within the central canal is also directly related to the rate of osteonal bone deposition and the size of osteoblasts;25) With the successive deposition of lamellae within developing osteons and the decrease in number and size of vessels within the central canal, the cellular functions of peripheral osteocytes rely more heavily on diffusion of nutrients and waste products through the tissue fluid of the canalicular system. Some investigators have suggested that the progressive decrease in osteonal vascularity creates a less favorable local environment in peripheral lacunae, causing metabolic abberations or cell death of their osteocytes. The micronecrotic area at the periphery of an old osteon 118 may in itia te the bone remodeling process by stimulating vascular ingrowth and necrotic bone resorption as a new resorption cavity is formed.1®

Capillaries within the central canals are surrounded by a continuous basal lamina. The endothelial cells comprising the capillary wall are fenestrated.24 The basal lamina apparently functions as a f ilt r a t io n stru ctu re during exchange between ca n a licu la r tissue flu id and plasma. By isotope tracer and morphometric techniques in the adult dog, the central canals were shown to occupy 1.3 to 1.5% o f c o rtic a l bone by volume. The to ta l c e llu la r volume o f c o rtic a l bone

(lacunar volume plus ca n a licu la r volume) was 4.2%?® (Baud and Vose have suggested a microcanalicular system which increases the c a n a lic u la r system volume by 100%, making the to ta l c e llu la r volume o f cortical bone approximately 7%.29>30 ) In one investigation, blood flow through cortical bone in the adult dog's femur was 3.7 ml/100

Gm/min, as compared to 19.6 m l/100 Gm/min in the femoral head, and 50.3 ml/100 Gm/min in the femoral neck.31

A rte ria l blood flows through the cortex from the endosteum to the periosteum under the driving pressure in the medullary arterioles.

Vascular patterns in this investigation and the studies of others 12,19 suggest that blood flow through venules arising from endosteal sinusoids and joining periosteal venules is also in the centrifugal d ire c tio n under normal re s tin g con dition s. Most o f the p u ls a tile driving force in marrow arterioles is probably dissipated by the distension of thin-walled arteriolar capillaries supplying endosteal sinusoids, and distension of the sinusoids themselves.®>32 Therefore, 119 a fa c to r la rg e ly responsible fo r the d ire c tio n and magnitude o f venous flow through the cortex is probably the venous pressure difference between the endosteal sinusoids and the extraosseous periosteal veins.

Under normal re stin g con dition s, marrow venous pressure is two to three times greater than extraosseous venousp r e s s u r e33 . The difference in pressures creates a ce n trifu g a l venous pressure gradient.

A ll marrow, c o r tic a l, and deep periosteal venous structures are valveless. The direction of venous blood flow through the cortex could easily be reversed (as Brookes,3 Rhinelander,? and W e ila n d ^ 3 have suggested) i f marrow venous pressure f e ll below periosteal venous pressure, or periosteal venous pressure became greater than marrow venous pressure. Muscular activity is believed to increase periosteal venous pressure.34 At these times cortical vessels may become engorged as venous outflow is impeded (or reversed) at the cortical bone-periosteal interface. Bone blood flow studies done in conscious dogs at rest and during exercise revealed a 50% increase in perfusion of femoral cortical bone after prolonged e x e r c i s e35 . The flow response was slow in developing and increased cortical blood flow persisted for one hour after physical exercise was terminated. Cortical blood flow did not decrease in itia lly as one would expect with an immediate increase in periosteal venous pressure. The slow response and persistent hyperemia, however, did suggest a metabolically induced stimulus (an increase in pC02 or decrease in p02 or pH).1 4 ,3 6 C o rtica l venous engorgement from impaired periosteal venous drainage would permit such alterations in the microenvironment. Increased vascular resistance in cortical venules leaving the endosteal sinusoids would also cause more blood to enter the marrow venous system. A ris e in extraosseous vascular pressure in diaphyseal emissary veins would increase venous drainage from the ends o f the marrow ca vity through metaphyseal and epiphyseal emissary veins. The dynamic effect adjacent muscles appear to have on periosteal vascular structures and cortical blood flow emphasizes the importance of existing alternate routes of venous drainage from the medullary cavity in maintaining adequate circulation in long bones. CHAPTER I I I

COMMUNICATION OF THE MARROW AND

CORTICAL BONE MICROCIRCULATION

121 ABSTRACT

A rte ria l supply to the marrow and c o rtic a l microvascular systems arose from separate arteriolar branches of medullary arteries.

Communication between the marrow and c o rtic a l venous m ic ro c irc u la tio n routinely occurred where portal venules arising from endosteal sinusoids traveled outward through the cortex to the periosteum. In general, both arterial and venous blood flow appeared to be in a centrifugal direction through the cortex.

Examples o f b id ire c tio n a l vascular patterns were id e n tifie d . Some resorption cavities of the inner cortex received a medullary arteriole and were drained by a venule which traveled back in to an endosteal sinusoid. Infrequently, an inner cortical arteriolar branch would re­ enter the marrow cavity and supply an endosteal sinusoid. In rare areas venule patterns within the cortex suggested some centripetal cortical venous drainage directly into the central medullary vein.

These areas were interpreted as intracortical capillary-venule anastomoses.

122 123

INTRODUCTION

Chapters I and II have described the general microvascular

patterns o f marrow and c o rtic a l bone as separate systems, fo r the sake

o f s im p lic ity . Whereas most o f the marrow is drained via the marrow venous system (central medullary vein and emissary veins),

intercommunication w ith the c o rtic a l venous sytem is also present.

Similarly, most of the cortical arterioles and capillaries drain into

the deep periosteal plexus. Within the cortex, however, occasional communication w ith the marrow venous system also occur. Branemark has

suggested that cortical bone and marrow should be regarded as an

anatomic and functional u n itJ The degree o f communication between the marrow and cortical microcirculations would support or discount the

validity of this concept.

The direction and characteristics of flood flow cannot be unequivocally reported from static vascular studies alone. Information

from two a d d itio n a l types o f studies—blood pressure measurements and v ita l microscopy o f marrow and c o rtic a l blood flo w —has been used in

this investigation to interpret observed vascular patterns. Blood pressure is greatest in medullary arterioles and capillaries, less in marrow venous stru ctu re s, and le a s t in periosteal v e i n s2 . »3 Blood has been observed to flow through arterioles at 1.0-1.5 mm/sec, capillaries at 0.5 mm/sec, venules a t 0.1-0.3 mm/sec, and sinusoids at 0-0.2 mm/sec. 124

MATERIALS AND METHODS

Twelve newborn puppies, 6 two-week-old puppies, 6 ten-week-old puppies, and 17 adult dogs were heparinized, anesthetized, catheterized, euthanized and perfused with orange or white pigmented

M icrofilr. (For details of the procedures, see Chapter I, Materials and Methods.)

Demonstration of the communication o f marrow and c o rtic a l microvascular structures was accomplished most consistently by prolonged antegrade arterial perfusion and gentle retrograde venous perfusion. Antegrade injection of M icrofilr was continued until only pure M icrofilr flowed from the venous catheter. The arterial catheter was plugged and a small amount of M icrofilr (determined solely by the amount of vascular resistance encountered) was injected retrograde, through the venous catheter. Specimens were harvested, processed, examined, and photographed as previously described. 125

RESULTS

The marrow and c o rtic a l m icro circu la to ry systems were found to be related to each other "in-parallel" rather than "in-series." Specific arterioles within the medullary cavity were responsible for providing vascular supply to cortical bone. Other arterioles gave off thin arteriolar capillary branches that emptied into sinusoids. Arterioles supplying sinusoids were not found to continue on to supply cortical bone, i.e ., an "in-series" relationship. When an "in-series" arrangement appeared to exist, close inspection revealed the arteriole passing behind the sinusoid into the cortex. Vascular structures traveling from sinusoids in the periphery of the marrow cavity

(endosteal sinusoids) into the cortex were frequently seen. These structures, however, had the morphological characteristics of venules, and differed significantly from the arteriolar capillaries that entered the sinusoids (Fig. 3.1). The venules were regarded as alternate sinusoidal drainage routes. Blood flowing from an endosteal sinusoid could enter the marrow venous drainage system (c o lle c tin g sinuses, central medullary vein and diaphyseal, metaphyseal, or epiphyseal emissary veins) or leave the sinusoid via the intracortical venules

(Fig. 3.2).

When located in the periphery of the medullary cavity, capillary- venule beds also exhibited an alternate route of venous drainage. In ad d itio n to the well-developed marrow venous drainage system of ven­ ules, collecting veins, and the central medullary vein, a few venules le ft the endosteal aspect of the capillary-venule bed and passed outwardly through the cortex to jo in the periosteal venous system Figure 3.1

Endosteal surface, mid-diaphysis; a d u lt dog. Small a r te rio la r c a p il­ lary (cap) supplying endosteal sinu- siuds from which a portal venule (ven) arises, art = arteriole.

Figure 3.2

Endosteal surface, mid-diaphysis; adult dog. Collecting sinuses and portal venules draining endosteal sinusoids. 127

FIGURE 3.1

FIGURE 3.2 128

(Figs. 3.3, 3.4). Arterial supply to the capillary-venules beds appeared to be re s tric te d to marrow c a p illa rie s ; no c o rtic a l capillaries entering the capillary-venule beds were identified.

In general, arterial and venous structures within cortical bone indicated a centrifugal, unidirectional route of blood flow. Several less frequently observed vascular patterns suggested a possible b i-d ire c tio n a l route o f blood flow between marrow and c o rtic a l bone microcirculatory systems. Arterioles within the medullary cavity entered cortical bone and returned again to the medullary cavity to jo in the marrow venous system.

Both u n id ire c tio n a l and b i-d ire c tio n a l m icrovascular systems were apparently involved in the bone remodeling process. Arising from arteries within the medullary cavity, cortical arterioles provided vas­ cular supply to developing resorption cavities. At the "cutter cone" head, a rte rio le s branched and doubled back w ith in the c a v ity as ven­ ules. At times the venules traveled outwardly through the cortex to link the marrow and periosteal microvascular systems. Bone re­ modeling in the outer cortex usually demonstrated this unidirectional vascular pattern (Figs. 3.5, 3.6). The venules from resorption cavi­ ties of the middle and inner cortex drained back into collecting veins or sinusoids within the medullary cavity (a bi-directional pattern of flo w ). Fewer and sm aller vessels were seen in the central canals of maturing osteons, indicating a "dropping out" of vascular structures as lamellae of osteonal bone fille d in the resorption cavity.

Nevertheless, the arteriovenous circuit from the medullary cavity, through cortical bone, and back into the medullary cavity was s till Figure 3.3

T ranscortical venous drainage o f a large capillary-venule bed at the periphery o f the marrow c a v ity ; ad ult dog.

Figure 3.4

Venules leaving the capillary- venule bed and entering the endos­ teal cortical surface; adult dog. 130

FIGURE 3 .3

FIGURE 3.4 Figure 3.5

Cortex (C) and endosteum (cu t sur­ face); adult dog. A resorption cavity (rc) being supplied by a cortical arteriole (art) from the medullary cavity, and drained by a cortical venule (ven) to the periosteum.

Figure 3.6

Cortex (cut surface); adult dog.

Medullary supply and periosteal drainage of a resorption cavity, art = cortical arteriole, ven = cortical venule. FIGURE 3 .5

FIGURE 3 .6 133 identifiable in some maturing osteons (Fig. 3.7).

Another type of inner cortical vascular circuitry was also iden­ tifie d . An arteriole or capillary, upon entering the endosteal corti­ cal surface, branched within the cortex. One intracortical capillary branch occasionally returned to the medullary cavity and entered an endosteal sinusoid, while other capillary branches passed outwardly through the cortex. The endosteal sinusoid apparently received vas­ cular supply from a cortical capillary, rather than from the arteriolar c a p illa rie s o f the marrow m icro circu la tio n (F ig. 3 .8 ).

In a few limited areas, intracortical venules drained directly into the central medullary vein. These venules did not appear to con­ tact periosteal vessels and were interpreted as representing an intra­ cortical capillary-venule anastomosis. In this instance, venous corti­ cal blood flow apparently avoided sinusoidal structures upon reentry into the medullary cavity (Fig. 3.9).

Interestingly, several isolated examples of sinusoids entrapped w ith in c o rtic a l bone were observed. The in tra c o rtic a l sinusoids were interposed between two or more venules. Since intracortical sinusoids were only seen on rare occasions and only when se le ctive venous in je c ­ tions were used, the possibility of arteriolar capillary supply to these sinusoids could not be determined (Fig. 3.10). Figure 3.7

Cortex (C) and endosteum (cut surface); adult dog. A resorption cavity being supplied by a cortical arteriole (art) and drained by a venule of an endos­ teal sinusoid (ven).

Figure 3.8

Cortex (C) and endosteum (cut sur­ face); adult dog. A cortical arteriole

(art) entering the cortex, branching and returning to medullary cavity to supply an endosteal sinusoid (EVS). 135

FIGURE 3 .7

FIGURE 3.8 Figure 3.9

Cortex (cut surface); adult dog.

Venules of an intracortical capil- lary-venule anastomosis draining directly into the central medullary vei n.

Figure 3.10

Cortex (cut surface); ad ult dog.

Isolated sinusoids entrapped within cortical bone. 137

FIGURE 3 .9

FIGURE 3.10 138

DISCUSSION

Information from many vascular studies has begun to slowly resolve

the question of whether separate microcirculations for marrow and cortical bone exist, or if circulation to marrow and cortical bone

involves one "interdependent and communicating" vascular system, as

Doan had suggested in 1921.5 From this investigation, both concepts appear to be partially correct.

In general, the marrow and c o rtic a l m icrovascular systems are

supplied by separate arterioles (marrow arterioles and cortical arterioles) arising from common parent vessels, the medullary arteries. In general, marrow and cortical bone are each drained by

separate venous systems (marrow via the central medullary and emissary

veins, and cortical bone via the periosteal veins) (Fig. 3.11). The

"in-parallel," rather than "in-series" concept of microcirculation in marrow and c o rtic a l bone has been supported by other anatomic and physiologic studies.6>7>8 This investigation, however, has shown that the marrow and cortical microvascular systems are not mutually exclusive.

The existence of intracortical communication of marrow and cortical microvascular structures has been questioned by some investigators.9^ 0 Intracortical communications which did not appear to join the periosteal vascular system were infrequently observed in this investigation. Under the driving force of the cortical arterioles and capillaries, arterial blood would flow into intracortical venules and venous blood would empty in to the low pressure marrow venous system. Venules from intracortical intercommunications may join Figure 3.11

Schematic drawing of unidirectional and bi-directional vascular intercommuni­ cations between marrow and c o rtic a l bone microcirculatory systems.

A = cortical arterioles

B = intersinusoidal communicating venule; drainage venule; portal venule

C = capillary-venule bed; portal venule

D = intracortical cappilary-venule anastomosis

E = sinusoidal system; collecting sinus

F = capillary-venule bed

G = deep periosteal venous plexus

H = superficial periosteum FIGURE 3.11 140 141

endosteal sinusoids or pass directly to join the central medullary

vein. This vascular pattern would permit the bi-directional flow of

blood through c o rtic a l bone, as observed by Branemark in v iv o .4

Communication between the marrow and c o rtic a l m icro circu la tio n s

also occurs in the deep periosteal plexus. Portal venules arising from

endosteal sinusoids usually radiate through the entire thickness of the

cortex and become continuous with deep periosteal venules. Normal flow

through the portal venules is probably c e n trifu g a l since marrow venous

pressure is greater than periosteal venous pressure. In th is au tho r's

opinion, the portal venules represent the vascular structures

responsible for permitting reversal of intracortical blood flow

whenever a s ig n ific a n t decrease in marrow venous pressure or increase

in periosteal venous presure occurs. Similar portal venules joining

endosteal capillary-venule beds with the deep periosteal plexus were

also present (Fig. 3.11, C). Because fatty marrow was infrequently

seen around the periphery of the medullary cavity, these portal venules

(leading from endosteal capillary-venule beds) were rarely observed.

Flow through the capillary-venule bed portal structures may only be centrifugal, since the driving force of capillary blood is greater than

the driving force of sinusoidal blood. In response to an appropriate

hematopoietic stimulus, conversion of the vascular structures of fatty marrow in to low pressure sinusoidal systems would enhance the

possibility of reversible flow within the portal venules.

Blood flowing from the medullary cavity, through cortical bone, and into the medullary cavity again (bi-directional flow) msy be a means by which the hematopoietic activity of marrow tissue is 142 regulated. Marrow tissu e must continuously respond to the ever changing demands of the body for erythrocytes, granulocytes and lymphocytes within a confined compartment. Increases in the volume of hematopoietic tissue require compensatory decreases in the volume of intramedullary vascular structures, cancellous bone, extracellular fluid or fatty marrow. Adipocytes constitute the most labile buffer.

Under a strong hematopoietic stimulus, all intramedullary fat may be mobilized within 48 hoursJ Ionized calcium, mobilized from cortical bone within central canals by erythropoietin or parathyroid hormone and returned to the medullary cavity via bi-directional vascular routes, may be the specific stimulus for the hematopoietic response. Calcium, erythropoietin, and parathyroid hormone all markedly stimulate mitosis in hematopoietic marrow tissue in vitro and in vivo.11*12,13 jhe stimulatory effect of calcium would presumably be greatest at the endosteal surface, where blood calcium le ve ls were h ig h e s t.^ In fa tty marrow areas, adipocytes c h a ra c te ris tic a lly occupy the central portions of the medullary cavity; hematopoietic tissue is dispersed around the periphery.

Communications between the marrow and c o rtic a l microvascular systems may prevent or decrease the amount of tissue necrosis during a temporary ischemic episode. Unfortunately, impaired circulation to the marrow often establishes a vicious cycle o f ischemia, edema and fib r o s is , increased marrow pressure, and increased vascular resistance. Increased vascular resistance worsens the ischemia, provoking further edema and ultimately results in the death of bone. In mature animals and man, obstruction to flow in 143 epiphyseal emissary veins appears to constitute the most serious threat to marrow and cancellous bone viability. Hips placed in particular positions of forced immobilization, and episodes of severe synovitis have caused Legg-Calve-Perthes disease (ischemic necrosis of the femoral head) in children.17,18,19 in an experimental study in young puppies, intracapsular tamponade of the hip jo in t with simultaneous blood flow measurements demonstrated an increase of 248% in femoral head marrow venous pressure and a 60% decrease in femoral head marrow blood flow .20 Also, osteonecrosis of the femoral head has been experimentally produced in young pigs and puppies by ligation of epiphyseal vessels.2^»22 Obstruction of epiphyseal emissary veins may cause less severe c lin ic a l problems in the a d u lt because o f the c o n tin u ity of epiphyseal and metaphyseal marrow venous systems.23*24

In ad d itio n to extraosseous venous fa c to rs , pathologic changes leading to osteonecrosis may be caused by extraosseous arterial factors, intraosseous arterial or venous factors, intraosseous extravascular factors and cellular cytotoxic factors.24

Commonly employed orthopedic practices compromise the c irc u la tio n of cortical bone to varying degrees. Cortical bone ischemia occurs beneath encircling Parham bands and along the contact surface of bone plates due to the obstruction of cortical venous drainage.2^ Damage to medullary vessels from fracture trauma is compounded by the use of intramedul1ary nails and pins. Intramedullary reaming and methylmetacrylate packing destroys the medullary source of cortical arterioles and the impermeable endosteal barrier of bone cement prevents the re-establishment of transcortical blood flow. When the 144 vascularity of bone is reduced, bone's resistance to infection is reduced, and post-operative sepsis may result.28

In addition to the role circulation undoubtedly plc^ys in the pathogenesis of certain orthopedic diseases, and the healing of fractures and other injuries to bone, circulation is intimately involved in the process of osteogenesis, the maintenance of bone v ita lity , the growth and remodeling of bone, and calcium-phosphorus metabolism. The necessity of adequate arterial supply and unrestricted venous drainage in accomplishing these diverse activities has made circulation in marrow and cortical bone a topic of sustained interest for many anatomists, physiologists, pathologists, and clinicians of radiology, internal medicine and surgery. LIST OF REFERENCES /

Preface

1. Ficat R. P., Arlet 0.: Ischemia and Necrosis of Bone. Baltimore, W illiams and Wilkens, 1980, pp. 29-30.

2. Hungerford D. S.: Pathogenetic Considerations in Ischemic Necro­ sis of Bone. Can. J. Surg. 24(6): 583-587, 1981

3. Rand J. A ., An K. N., Chao E. Y. S ., K elly P. J .: A Comparison of the E ffects o f Open Intram edullary N ailing and Compression Plate Fixation on Fracture Site Blood Flow and Fracture Union. J. Bone Joint Surg. 63-A:427-442, 1981.

4. Robbins S. L.: Pathologic Basis of Disease. Philadelphia, W.B. Saunders, Co., 1974, pp. 1436-1440.

5. Dubielzieg R. R., Biery D. N., Brodey R. S.: Bone Sarcomas Asso­ ciated With Multifocal Medullary Bone Infarction in Dogs. J. Am. Vet. Med. Assoc., 179(1):64-68, 1981.

6. Johnston A. D.: Pathology of Metastatic Tumor in Bone. Clin. Orthop. 73:8-32, 1970.

7. Aegerter E. and Kirkpatrick J. A.: Orthopedic Diseases: Physiology Pathology, Radiology, 4th ed. P hiladelphia, W. B. Saunders Co., 1975, pp. 430-433.

8. Suramo I . , Puranen J ., Heikkinen E., Vuorinen P.: Disturbed Patterns of Venous Drainage of the Femoral Neck in Perthes' Disease. J. Bone Joint Surg. 56-B:448-453, 1974

9. Heikkinen E. S., Lanning P ., Suramo I . , Purannen J .: The Venous Drainage of the Femoral Neck as a Prognostic Sign in Perthes' Disease. Acta Orthop. Scand. 51:501-503, 1980

10. Green N. E., Guffin P. P.: Intra-osseous Venous Pressure in Legg- Perthes' Disease. J. Bone Joint Surg. 64-A:666-671, 1982

145 Ohtani 0., Gannon B., Ohtsuka A., Murakami T .: The Microcircu­ la tio n o f Bone and E specially o f Bone Marrow as studied by Scan ning Electron Microscopy of Vascular Casts--A Review. Scan. Electron Micros. 1:427-434, 1982. 147

Chapter I

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2. Trueta J., Harrison M. H. M.: The Normal Vascular Anatomy of the Femoral Head in Adult Man. J. Bone Jo in t Surg. 35-B:442-461, 1953.

3. Tavassoli M., Yoffey J. M.: Bone Marrow: Structure and Function. New York, Alan R. Liss, In c ., 1983, pp. 65, 84-85, 82, 80.

4. Spalteholz W.: Uber das Durchsichttigmachen von Menschlichen und Tierischen Praparaten. Leipzig, Verlag Von S. Hirzel, 1911, pp. 43-48.

5. Kaderly R. E., Anderson B. G., Anderson W. D .: Intracapsular and Intraosseous Vascular Supply to the Mature Dog's Coxofemoral Joint. Am. J. Vet. Res. 44(10):1805-1812, 1983.

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8. Yoffey J. M.: A Note on the Thick-Walled and Thin-Walled Arteries o f Bone Marrow. J. Anat. 96:425, 1962.

9. Tavassoli M.: Arterial Structure of Bone Marrow in the Rabbit With Special Reference to the Thin-Walled Arteries. Acta Anat. 90(4):608-616, 1974.

10. Zamboni J ., Pease D. C .: The Vascular Bed o f Red Bone Marrow. J. Ultrastr. Res. 5:65-85, 1961

11. DeBruyn P. P. H ., Michelson S., Thomas T. B.: The M igration o f Blood Cells o f the Bone Marrow Through the Sinusoidal W all. J. Morph. 133:417-438, 1971.

12. DeBruyn P. P. H., Breen P. C., Thomas T. B .: The M icro circu la tio n o f the Bone Marrow. Anat. Rec. 168(1):55-68, 1970. 148 13. Ohtani 0 ., Gannon B ., Ohtsuka A ., Muratami T .: The M icrovasculature of Bone and Especially of Bone Marrow as Studied by Scanning Elec­ tron Microscopy o f Vascular Casts—A Review. Scan. Electron Microsc. 1:427-434, 1982.

14. Lopez-Curto J. A., Bassingthwaighte J. B., Kelly P. J.: Anatomy of the Microvasculatrue of the Tibial Diaphysis of the Adult Dog. J. Bone Joint Surg. 62-A:1362-1369, 1980.

15. Brooks M., Harrison R. G.: The Vascularization of the Rabbit Femur and Tibiofibula. J. Anat. 91:61-72, 1957.

16. Branemark P. I . : V ita l Microscopy o f Bone Marrow in Rabbit. Scand. J. Lab Invest. 11, Suppl. 38, 1-82, 1959.

17. Lichtman M. A.: The Ultrastructure of the Hemopoietic Environment o f the Marrow: A Review. Exp. Hematol. 9 (4 );391-410, 1981.

18. Martens R. J., Auer J. A.: Hematogenous Septic A rhtritis and Osteo­ myelitis in the Foal. Proc. 6th Meeting of the Am. Assoc. Eg. Pr?ct. Anaheim , C a lifo rn ia , 1980, pp. 47-63.

19. Ogden J. A., Lister G.: The Pathology of Neonatal Osteomyelitis. Pediatrics 55(4):474-478, 1975.

20. Tavassoli M., Yoffey J. M.: Bone Marrow: Structure and Function. New York, Alan R. L iss, In c ., 1983, pp. 70-71.

21. Tondevold E., Eliasen P.: Regional Vascular Volumes and Dynamic Haematocrit Compared to Regional Perfusion in Canine Cancellous and C ortical Bone. Acta Orthop. Scand. 53(2):197-203» 1982.

22. Kinosita R ., Ohno S ., Bierman H. R .: Regenerating Bone Marrow in Situ. Fed. Proc. 16:1-7, 1957.

23. Yoffey J. M.: Lymphocyte Loading o f Bone Marrow Sinusoids. Adv. Microcirc. 7:49-67, 1977.

24. Tavassoli M., Aoki M.: Migration of Entire Megakarocytes Through the Marrow-Blood Barrier. Br. J. Haematol. 48:25-29, 1981.

25. McCuskey R. S ., McCluggage S. G., Younker W. J . : ' Microscopy o f Living Bone Marrow in S itu . Blood. 39:87-95, 1971.

26. Yoffey J. M., Hudson G., Osmond D. G .: The Lymphocyte in Guinea- Pig Bone Marrow. J. Anat. 99:841-860, 1965.

27. Branemark P. I . : Bone Marrow Microvascular S tructure and Fucntion. Adv. M ic ro c irc . 1:1-65, 1968.

28. Rappaport H ., Braum M ., H o rre ll J. B .: Bone Marrow Embolism. Am. J . Pathol. 27:407-433, 1951. 149 29. McCarthy P. G., Schmookler B. M ., Pierce L. E .: Fatal Bone-Marrow Pulmonary Emboli in M u ltip le Myeloma. Ann. In te rn . Med. 86:317-318, 1977.

30. Modig J .: Medullary Fat Embolization During Total Hip Replacement Surgery: A Preliminary Report. Injury 5:161-164, 1973.

31. Steinbach H. L., Jergensen F., G ilfillan R. S., Petrakis N. L.: Osseous Phlebography. Surg. Gynec. Obstet. 104:215-226, 1957.

32. Schobinger R. A., Ruzicka F. F.: Transosseous Phlebography: Gene­ ral Principles in Vascular Roentgenology: Arteriography, Phlebo­ graphy, Lymphography. New York, The MacMillan Co., 1964, p. 46.

33. Weiss L: Transmural Cellular Passage in Vascular Sinuses of Rat Bone Marrow. Blood 36:189-208, 1970.

34. Campbell F. R.: Ultrastructural Studies of Transmural Migration of Blood C ells in the Bone Marrow o f Rats, Mice, and Guinea Pigs. Am. J. Anat. 135:521-536, 1972.

35. Becker R. P., DeBruyn P. P. H.: The Transmural Passage of Blood Cells into Myeloid Sinusoids and the Entry of Platelets into Sinu­ soidal Circulation: A Scanning Electron Microscopic Investigation. Am. J. Anat. 145:183-206, 1976.

36. Hudson G., Yoffrey J. M.: Reticulo-endothelial Cells in the Bone Marrow of the Guinea-Pig. J. Anat. 97:409-416, 1963.

37. Michelsen K.: Pressure Relationships in the Bone Marrow Vascular Bed. Acta Physio. Scand. 71:16-29, 1967.

38. K inosita R ., Ohno S ., Bierman H. R .: Observations on Regenerating Bone Marrow Tissue In S itu . Proc. Am. Assoc. Cancer Res. 2:125, 1956.

39. Robbins S. L . , Angell M., Kumar V.: The Musculoskeletal System: Bones in Basic Pathology. P hiladelphia, W. B. Saunders Co., 1981, pp. 630-631.

40. Aegerter E ., K irk p a tric k J. A .: General Considerations o f Tumors, in Orthopedic Diseases: Physiology, Pathology, Radiology. Phila­ delphia, W. B. Saunders Co., 1975, pp. 472-474.

41. Shaw N. E.: Observations on the Physiology of the Circulation in Bones. Ann. R. C o ll. Surg. Eng. 35:214-233, 1964.

42. Weiss L: The S tructure o f Bone Marrow. J. Morph. 117:467-538, 1965.

43. Shim S. S.: Physiology of Blood Circulation of Bone. J. Bone Joint Surg. 50-A:812-824, 1968. 150 44. Gross P. M., Heistad D. D ., Marcus M. L .: Neurohumeral Regulation of Blood Flow to Bones and Marrow. Am. J. Physio. 237(4) 440-448, 1979. 45. Tondevold E ., Eliasen P.: Bone Blood Flow in Conscious Dogs at Rest and During Exercise. Acta Orthop. Scand. 54(l):53-57, 1983.

46. Brookes M .: Blood Flow Rate in Compact and Cancellous Bone, and Bone Marrow. J. Anat. 101:533-541, 1967.

47. Singh M., Brookes M.: Bone Growth and Blood Flow A fte r Experimen­ tal Venous ligation. J. Anat. 108:315-322, 1971.

48. Gross P. M., Marcus M. L ., Heistad D. D .: Current Concepts Review: Measurement o f Blood Flow to Bone and Marrow in Experimental Animals by Means of the Microsphere Technique. J. Bone Joint Surq. 63-A: 1028-1031, 1981.

49. Tondevold E ., Eliasen P.: Blood Flow Rates in Canine C ortical and Cancellous Bone Measured with 99jcm- Labelled Human Albumin Micro­ spheres. Acta Orthop. Scand. 53:7-11, 1982.

50. De Bruyn P. P. H.» Thomas T. B ., Michelson S .: Fine S tructure o f the Vascular Components o f the Guinea Pig Bone Marrow. Anat. Rec. 154:499, 1966. ------

51. Hudson G., Yoffey J. M.: Ultrastructure of Reticuloendothelial Ele­ ments in Guinea-Pig Bone Marrow. J. Anat. 103:515-525, 1968.

52. Michelsen K.: Determination in Inulin, Albumin, and Erythrocyte Spaces in the Bone Marrow o f Rabbits. Acta P hysiol. Scand. 77: 28-35, 1969. 151 Chapter I I

1. Johnson R. W.: A Physiological Study of the Blood Supply of the Diaphysis. J. Bone Joint Surg. 9:153-184, 1927.

2. Brookes, M., Harrison R. G.: The Vascularization of the Rabbit Femur and Tibio fibula. J. Anat. 91:61-69, 1957.

3. Brookes M.: The Blood Supply o f Bone, in Clark J. M. P. (ed): Modern Trends in Orthopaedics, Washington, Butterworth and Co., 1964, vol 4, pp. 91-125.

4. Crock H. V.: The Blood Supply o f the Lower Limb Bones in Man. Edinburgh and London, E & S Livingstone L td ., 1967, pp. 4-15.

5. Doan C. A.: The Circulation of the Bone Marrow, in Contributions to Embryology XIV, Washington, Carnegie Institute of Washington, 1922, p. 29.

6. Kelly P. J., Janes J. M.: Microangiographic and Histologic Studies o f Vascular Anatomy o f the Femur and T ib ia D istal to a Femoral and Iliac Arteriovenous Fistula in Dogs. Anat. Rec. 162:255-267, 1968.

7. K elly P. J . : Comparison o f Marrow and C ortica l Bone Blood Flow by 125i-Labeled 4-Iodoantipyrine (I-A p) Washout. J. Lab. C lin . Med. 81:497-505, 1973.

8. Lopez-Curto J. A., Bassingthwaighte J. B., Kelly P. J.: Anatomy of the Microvasculature of the Tibia! Diaphysis of the Adult Dog. J. Bone Joint Surg. 62-A.-1362-1369, 1980.

9. DeBruyn P. P. H ., Breen P.C., Thomas T. B ., The M icro c irc u la tio n o f the Bone Marrow. Anat. Rec. 168:55-68, 1970.

10. Ohtani 0., Gannon B., Ohtsuka A., Murakami T .: The Microvasculature of Bone and Especially of Bone Marrow as Studied by Scanning Elec­ tron Microscopy o f Vascular Casts: A Review. Scan. Electron Mic- rosc. 1:427-434, 1982. 11. Perris A. D., Whitfield J. F., Rixon R. H.: Stimulation of Mitosis in Bone Marrow and Thymus o f Normal and Irra d ia te d Rats by Divalent Cations and Parathyroid Extract. Radiat. Res. 32:550-563, 1967.

12. Morton H. J .: Effect of Calcium on Bone Marrow Mitosis In Vitro. Proc. Soc. Exp. Biol. Med. 128:112-116, 1968.

13. Hunt N. H., Perris A. D.: Erythropoietin-Induced Changes in Plasma Calcium and Bone Marrow Mitosis in the Rat. J. Endocrinol. 56: 47-57, 1973. 152 14. Tavassoli M., Yoffey J. M.: Bone Marrow: Structure and Function. New York, Alan R. L iss, In c ., 1983, p. 69.

15. Wilkes C. H., Visscher M. B.: Some Physiological Aspects of Bone Marrow Pressure. J. Bone Joint Surg. 57-A:49-57, 1975.

16. Hungerford D. S .: Bone Marrow Pressure, Venography, and Core Decom­ pression in Ischemic Necrosis of the Femoral Head, in The Hip, Proc. 7th Meeting o f the Hip Soc., 1979, pp. 218-237.

17. Gore D. R.: Iatrogenic Avascular Necrosis of the Hip in Young Children. J. Bone Joint Surg. 56-A:493-502, 1974.

18. Schoenecker P. L., Bitz D. M., Whiteside L. A.: The Acute Effect of Position of Immobilization on Capital Femoral Epiphyseal Blood Flow. J. Bone Joint Surg. 60-A:899-904, 1978.

19. Jacobs B. W.: Synovitis of the Hip in Children and Its Significance. Pediatrics 47:558-566, 1971.

20. Launder W.J.. Hunqerford D. S ., Jones L. H.: Hemodynamics o f the Femoral Head. J. Bone J o in t Surg. 63-A:442-448, 1981.

21. S a lte r R. B ., B ell M.:The Pathogenesis of Deformity in Legg- Perthes Disease--An Experimental Investigation. The Can. Orthop. Assos., Proc. of the 23rd Ann. Meeting, Montreal, 1967.

22. Sanchis M,. Zahir A., Freeman M. A. R.: The Experimental Simulation of Perthes Disease by Consecutive Interruptions of the Blood Supply to the Capital Femoral Epiphysis in the Puppy. J. Bone Joint Surg. 55-A:335-342, 1973.

23. Arnoldi, C. C., Linderholm H., Mussbichler H.: Venous Engorgement and Intraosseous Hypertension in Osteoarthritis of the Hip. J. Bone Joint Surg. 54-B:409-421, 1972.

24. Ogden J. A .: Changing Patterns o f Proximal Femoral V ascula rity. J. Bone Joint Surg. 56-A:941-950, 1974.

25. Hungerford D. S.: Pathogenetic Considerations in Ischemic Necrosis o f Bone. Can. J. Surg. 4(6):583-587, 1981.

26. Rhinelander F. W.: The Normal Microcirculation of Diaphyseal Cortex and Its Response to Fracture. J. Bone Joint Surg. 50-A:784-800, 1968.

27. Sorensen W. G., Bloom J. D., K elly P. J .: The E ffects o f Intrame- dullary Methylmethacrylate and Reaming on the Circulation of the Tibia After Osteotomy and Plate Fixation in Dogs. J. Bone Joint Surg. 61-A:417-242, 1979.

28. Brookes M.: Circulatory Depression in Bone After Acrylic Implanta­ tion. Clin. Orthop. 107:274-276, 1975. 153

29. Vose G. P.: Fine S tructure o f Bone as Seen on Fracture and Cleavage Planes by Electron Microscopy. Anat. Rec. 145:183-191, 1963.

30. Baud C. A.: Submicroscopic Structure and Functional Aspects of the Osteocyte. Clin. Orthop. 56:227-236, 1968.

31. Tondevold E., Eliasen P.: Blood Flow Rates in Canine C ortical and Cancellous Bone Measured With 99Tcni-Labelled Human Albumin Micro­ spheres. Acta Orthop. Scand. 53:7-11, 1982.

32. Tavassoli M., Yoffey J. M.: Bone Marrow: Structure and Function. New York, Alan R. Liss, Inc. 1983, pp. 70-71.

33. Michelsen K .: Pressure R elationships in the Bone Marrow Vascular Bed. Acta Physio. Scand. 71:16-29, 1967.

34. Shaw N. E.: Observations on the Physiology of the Circulation in Bones. Ann. R. C o ll. Surg. Eng. 35:214-233, 1964.

35. Tondevold E., Eliasen P.: Bone Blood Flow in Conscious Dogs at Rest and During Exercise. Acta Orthop. Scand. 54(1):53-57. 1983.

36. Brookes M.: Blood Flow Rate in Compact and Cancellous Bone and Bone Marrow. J. Anat. 101:533-541, 1967. 154 Chapter I I I

1. Branemark P. I . : Bone Marrow Microvascular S tructure and Function. Adv. M icro circ. 1:1-65, 1968.

2. Michelsen K.: Pressure Relationships in the Bone Marrow Vascular Bed. Acta Physio Scand. 71:16-29, 1967.

3. Jee W. S. S.: The Skeletal Tissues, in WeissL.: Histology, 5th ed., New York, McGraw-Hill, 1983, pp. 223-225.

4. Branemark P. I . : V ita l Microscopy o f Bone Marrow in Rabbit. Scand. J. Lab. In ve st. 11, Supp;. 38, 1-82, 1959.

5. Rhinelander F. W.: C ircu la tio n in Bone, in Bourne C. (ed): The Biochemistry and Physiology o f Bone, 2nd ed. New York and London, Academic press, In c ., 1972, v o l.2, pp. 14-15.

6. Branemark P. I . : V ita l Microscopy o f Bone Marrow in Rabbit. Scand. J. Clin. Lab. Invest., Suppl. 11, 38:1-82, 1959.

7. Rhinelander F. W.: The Normal Microcirculation of Diaphyseal Cortex and Its Response to Fracture. J. Bone Joint Surg. 50-A:784-800, 1968.

8. K e lly P. J .: Anatomy, Physiology, and Pathology o f the Blood Supply of the Human Tibia. J. Bone Joint Surg. 50-A:766-782, 1968.

9. Nelson G. E., K elly P. J ., Peterson L. F. A ., Janes J. M.: Blood Supply of the Human Tibia. J. Bone Joint Surg. 42-A:625-636, 1960.

10. DeBruyn P. P. H., Breen P. C., Thomas T. B.: The M icro circu la tio n o f the Bone Marrow. Anat. Rec. 168:55-68, 1970.

11. Trias A., Ferey A.: Cortical Circulation of Long Bones. J. Bone Joint Surg. 61-A:1052-1059, 1979.

12. Lopez-Curto J. A., Bassingthwaighte J. B., Kelly P. J.: Anatomy of the Microvasculature of the Tibia! Diaphysis of the Adult Dog. J. Bone Joint Surg. 62-A:1362-1369, 1980.

13. Weiland A. J ., Berggren A., Jones L.: The Acute Effects of Blocking Medullary Blood Supply on Regional C ortical Blood Flow in Canine Ribs as Measured by the Hydrogen Washout Technique. C lin. Orthop. 165:265-272, 1982.

14. Shim S. S.: Physiology of Blood Circulation of Bone. J. Bone Joint Surg. 50-A:812-824, 1968. 155 15. Jee W. S. S.: The Influence of Reduced Local Vascularity on the Rate of Internal Reconstruction in Adult Long Bone Cortex, in Frost (ed): Bone Biodynamics. Boston, L it t le Brown, 1964, pp. 259-277.

16. Brookes M., Singh M.: Bone Blood pH and Gas Tensions A fte r Femoral Vein Ligation. Surg. Gynecol, Obstet. 135:873-876, 1972.

17. Mclnnis J. C., Robb R. A., Kelly P. J.: The Relationship of Bone Blood Flow, Bone Tracer Deposition and Endosteal New Bone Formation. J. Lab. Clin. Med. 96(3):511-522, 1980.

18. Trueta J .: The Dynamics of Bone Circulation, in Frost (ed): Bone Biodynamics. Boston, L it t le Brown, 1964, pp. 245-258.

19. Ohtani 0., Gannon B., Ohtsuka A., Murakami T.: The Microvasculature o f Bone and E specially o f Bone Marrow as Studied by Scanning Elec­ tro n Microscopy o f Vascular Casts: A Review. Scan. Electron Microsc. 1:427-434, 1982.

20. Morris M. A., Kelly P. J.: Use of Tracer Microspheres to Measure Bone Blood Flow in Conscious Dogs. Calcif. Tissue Int. 32(l):69-76, 1980.

21. Currey J. D.: Differences in the Blood Supply of Bones of Different Histological Types. Q. J. Microsc. Sci. 101:351-370, 1960.

22. Vasciaveo F ., B a rto li E .: Vascular Channels and Resorption C avities in Long Bone Cortex. The Bovine Bone. Acta Anat. 47:1-33, 1961.

23. K elly P. J . : Comparison o f Marrow and C ortica l Bone Blood Flow by l 23I-Labeled 4-Iodoantipyrine (I-A p) Washout. J. Lab. and C lin . Med. 81:497-505, 1973.

24. Cooper R. B ., Milgram J. W., Robinson R. A .: Morphology o f the Os­ teon: An Electron Microscopic Study. J. Bone Joint Surg. 48-A: 1239-1271, 1966.

25. Marotti G., Zallone A. Z.: Changes in the Vascular Network During the Formation of Haversian Systems. Acta Anat. 106:84-100, 1980.

26. Branemark P. I . : Bone Marrow M icrovascular S tructure and Function. Adv. M ic ro c irc . 1:1-65, 1968.

27. Cohen J ., Harris W. H.: The Three Dimensional Anatomy of Haversian Systems. J. Bone Jo in t Surg. 40-A:419-434, 1958.

28. Morris M. A., Lopez-Curto J. A., Hughes S. P. F., An K, N, Bassingwaighte J. B., Kelly P. J.: Fluid Spaces in Canine Bone and Marrow. Microvasc. Res. 23(2):188-200, 1982.