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1986 Experimental Hemarthrosis in Rhesus Monkeys: Light Microscopic, Scanning and Transmission Electron Microscopic, Biochemical, Metabolic and Morphometric Analyses. David Henry Zeman Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Zeman, David Henry, "Experimental Hemarthrosis in Rhesus Monkeys: Light Microscopic, Scanning and Transmission Electron Microscopic, Biochemical, Metabolic and Morphometric Analyses." (1986). LSU Historical Dissertations and Theses. 4276. https://digitalcommons.lsu.edu/gradschool_disstheses/4276
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T TA/f.T Dissertation U 1V 11 Information Service University Microfilms International A Bell & Howell Information Company 300 N. Zeeb Road. Ann Arbor, Michigan 48106 8629210
Zeman, David Henry
EXPERIMENTAL HEM ARTHROSIS IN RHESUS MONKEYS: LIGHT MICROSCOPIC, SCANNING AND TRANSMISSION ELECTRON MICROSCOPIC, BIOCHEMICAL, METABOLIC AND MORPHOMETRIC ANALYSES
The Louisiana State University and Agricultural and Mechanical Col. Ph.D. 1986
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University Microfilms International EXPERIMENTAL HEMARTHROSIS IN RHESUS MONKEYS: LIGHT MICROSCOPIC, SCANNING AND TRANSMISSION ELECTRON MICROSCOPIC, BIOCHEMICAL, METABOLIC AND MORPHOMETRIC ANALYSES
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Interdepartmental Program ' in Veterinary Medical Sciences
Option: Veterinary Pathology
by David Henry Zeman B.S., North Dakota State University, 1976 D.V.M., Oklahoma State University, 1980 May 1986 ACKNOWLEDGEMENTS
I am sincerely appreciative of the many individuals who have played roles in my training as a veterinary pathologist at Louisiana State University. My major professor, Dr. Don Roberts, provided a perfect blend of guidance and freedom which motivated my professional development and encouraged freethinlcing. I am grateful for his efforts and will miss his friendship as I leave. I am also thankful to other members of my graduate committee,
Dr.s W.G. Henlc, H.W. Casey, K.A. Gossett and G.B. Baskin.
This project would not have been possible without the collaboration of the Delta Regional Primate Research
Center of Tulane University, Covington, LA and the
Departments of Pathology and Orthopedics of the LSU-Medical
Center in New Orleans. Both institutions provided experimental animals and facilities for which I am indeed grateful. A special thanks to: Dr.s Baskin and Watson who assisted during the injection and collection procedures at the primate center; Dr.s Neuman and Eggen who allowed us to work with them during sacrifice times at the Department of
Pathology, LSU-Medical Center; and Dr.s Shoji and Miwa who guided me through the biochemical and metabolic procedures at the Orthopedic Research Laboratory of the Department of
Orthopedics, LSU-Medical Center.
I am grateful to the University, the School of Veterinary Medicine and the Department of Veterinary
Pathology for providing my stipend during this work.
Thanks also to the many veterinary pathologists who played important roles in my morphologic training: Dr.s Roberts,
Casey, Cho, Snider, J. Turk, P. Turk, Taylor, Miller,
Williams, Baskin, Hodgin, Lozano, Gossett, Gaunt and Baker.
Many thanks to department technical staff: the
Histology Lab for tissue processing (Lynn Montgomery,
Cheryl Crowder, C.A. Green and Del Phillips); Cheryl Crowder for doing Safranin-0 special stains; and Mary Bowen and
Andy Smith for EM tissue sectioning.
Thanks for technical assistance outside of the department includes Michael Broussard who did the diagrams and graphics in this manuscript and Harry Cowgill who provided photographic material for seminars; both are from the Department of Instructional Resources. And thanks to
Sherry Gibson and Laura Younger from the Electron
Microscopy Center-S.V.M. for advice and assistance during scope operation.
Most importantly, I thank Colleen, Erik and Angela for their patience, understanding and love during this work.
They have been my motivation and make all of this seem worthwhile.
iii TABLE OF CONTENTS
Page
Acknowledgements...... ii
Table of Contents...... iv
List of Figures ...... vi
List of Tables...... ix
Abstract...... x
Introduction and Objectives...... 1
Literature Review
Anatomy, Physiology and Pathology of Synovial Joints....6
Hemarthrosis...... 39
Calcium Pyrophosphate Dihydrate Deposition Disease..... 50
Materials and Methods...... 61
Results
Morphologic Observations...... 77
Biochemical Analyses of Articular Cartilage...... 106
Metabolic Analyses of Articular Cartilage...... 106
M o r p h o m e t r i c A n a l y s e s ...... 109
Discussion
Morphologic Interpretation...... 114
Biochemical, Metabolic and Morphometric Studies...... 122
Hemarthrosis and CPPD Crystals in Rhesus Monkeys...... 125
Conclusions...... 129
Bibliography...... 134
Appendix...... 156
iv TABLE OF CONTENTS (continued)
Vita......
Approval Sheets
v LIST OF FIGURES
Page
Figure 1. Mankin osteoarthritis grading system...... 66
Figure 2. Isotope labeling procedure for articular
cartilage...... 69
Figure 3. Initial biochemical processing of cartilage..69
Figure 4. Procedure for total protein assay of
articular cartilage...... 71
Figure 5. Procedure for hexosamine assay of cartilage..71
Figure 6. Procedure for hydroxyproline assay of
articular cartilage...... 73
Figure 7. Cytoplasmic vacuoles in principal
chondrocytes (TEM)...... 80
Figure 8. Cytoplasmic processes in principal
chondrocytes (TEM)...... 81
Figure 9. Myelin figures in principal
chondrocytes (TEM)...... 82
Figure 10. Lipid droplets and siderosomes in principal
chondrocytes (TEM)...... 83
Figure 11. Normal variations in control
chondrocytes (TEM)...... ,...... 85
Figure 12. Cartilage crystal (TEM)...... 86
Figure 13. Femoral and tibial articular surfaces (TEM)..88
Figure 14. Hyperplastic synovial membrane from a
principal joint (LM)...... 89 Figure 15, Fibrinohemorrhagic material trapped
between villi (LM)...... 91
Figure 16. Overview of control synovial membrane (TEM).,93
Figure 17. Typical cytoplasmic components of normal
type A and B synoviocytes (TEM)...... 94
Figure 18. Overview of hyperplastic synovial membrane
from a principal joint (TEM)...... 95
Figure 19. Vacuolated type A synoviocytes from a
principal joint (TEM)...... 97
Figure 20. Whorled membranous bodies in a type A
synoviocyte (TEM)...... 98
Figure 21, Control perimeniscal synovial membrane (SEM).99
Figure 22. Control synovial membrane from a flat,
cellular area (SEM) ...... 101
Figure 23. Fat pad synovial membrane (SEM)...... 102
Figure 24. Hyperplastic principal synovial
membrane (SEM)...... 103
Figure 25. Red cells on synovium surface of a
principal joint...... 104
Figure 26, Results of cartilage total protein assay....107
Figure 27. Results of cartilage hexosamine assay...... 107
Figure 28. Results of hydroxy proline assay...... 108
Figure 29. Results of morphometric evaluation of
synovial membrane cellularity...... 110
vii Figure 30. Results of morphometric evaluation of
synovial membrane intima thickness...... 110
Figure 31. Principal and control cartilage (LM)...... 112
Figure 32. Results of morphometric evaluation of
cartilage cellularity...... 113
Figure 33. Results of morphometric evaluation of
average chondrocyte lacuna area...... 113
viii LIST OF TABLES
Pag-e
Table 1. Mankin Osteoarthritis Grade...... 157
Table 2. Cartilage Total Protein Assay...... 158
Table 3. Cartilage Hexosamine Assay...... 159
Table 4. Cartilage Hydroxyproline Assay...... 160
Table 5. Cartilage Protein Production ...... 161
Table 6. Cartilage Proteoglycan Production...... 162
Table 7. Cartilage Collagenous Protein Production.... 163
Table 8. Secreted Protein Production...... 164
Table 9, Secreted Proteoglycan Production...... 165
Table 10. Synovial Membrane Cellularity...... -....166
Table 11. Thickness of Synovial Membrane Intima...... 167
Table 12. Articular Cartilage Cellularity...... 168
Table 13. Average Lacuna Area of Articular Cartilage..169
Table 14. Matrix Cell Area Ratios of Articular
Cartilage...... 170
Table 15. Percentage of Articular Cartilage Area
Occupied by Lacunae...... 171
Table 16. Subchondral Bone Area Expressed as
Percentage of Total Subchondral Area...... 172
Table 17. Osteocyte Lacuna Area of Subchondral Bone
Expressed as Percent of Trabeculae Area 173
ix ABSTRACT
Hemarthrosis in rhesus monkeys was studied to: 1) provide information regarding the possible relationship between traumatic hemarthrosis and naturally occurring calcium pyrophosphate dihydrate-deposition disease (CPPD-
D D ) in rhesus monkeys, 2 ) fill a void in the literature regarding experimental hemarthrosis in primates, 3) and study the early changes that occur in blood-induced cartilage destruction by adding biochemical, metabolic and morphometric analyses to the traditional morphologic evaluations. Three ml of autologous blood were injected into the left knee of 16 anesthetized monkeys; the right • knee was an untreated control. Monkeys were sacrificed at
7 days, 2, 3, and 6 months post injection (PI). Synovial membrane and articular cartilage were evaluated by light microscopy, and scanning and transmission electron microscopy (EM). Articular cartilage was further analyzed by biochemical, metabolic and morphometric procedures.
There was a hyperplastic and inflammatory reaction in the synovium at 7-days PI, which had r e s o l v e d by 2 months
PI. Synoviocytes in the 7-day group contained numerous cytoplasmic vacuoles and prominent microplicae,
Erythrophagocytosis by synoviocytes was observed by light microscopy and confirmed by transmission EM. Scanning EM revealed another possible route of blood removal from
x joints; red cells were frequently interposed between synoviocytes, suggesting movement through the synovial intima. Mild degenerative changes in superficial chondrocytes included increased numbers of myelin figures and cytoplasmic vacuolation.
There was an overall significant decrease in cartilage proteoglycan content of principal joints as well as increased collagenous protein production. The cartilage of principal joints was hypercellular relative to controls.
No differences were found between principal and control joints in regards to crystals within the pericellular and territorial matrix.
In conclusion: 1) Rhesus monkeys reacted to hemarthrosis similar to dogs and rabbits, resulting in mild morphologic changes with resolution by 2-months PI. 2)
Early changes in blood-induced cartilage destruction in rhesus monkeys is apparently related to noxious influences on chondrocytes and interference with their ability to maintain the cartilage matrix. 3) Experimental hemarthrosis failed to produce CPPD crystal s. The joint insult produced by a single episode of hemarthrosis may not have been prolonged or severe enough for proposed crystallization mechanisms to act. INTRODUCTION AND OBJECTIVES
Hemarthrosis, by definition, is the "extravasation of c £ blood into a joint or its synovial cavity". Interest in hemarthrosis has largely come from the condition in hemophiliacs. Although hemophilia has been a medical condition recognized for several hundred years, it was not until the 19th century that it was associated with
1ort OIL hemarthrosis. ' In human hemophiliacs, multiple hemarthrotic episodes eventually produce a conditon known as hemophilic arthropathy. In this condition, chronic proliferative changes in the synovial membrane and eventual degenerative changes in the articular cartilage create a severe debilitating arthropathy in affected individuals.11» 253,193
Consequently, much of the experimental work in hem arthrosis has been directed towards the pathogenesis of chronic hemophilic arthropathy with experiments being designed to reproduce that condition.^ 2,216,220 Rabbits became the most utilized experimental animal in hemarthrotic 91 7 0 90 Since Swanton reported hemophilic arthropathy in 259 dogs to be essentially identical to that of man, 112 hemarthrosis experiments have also utilized dogs.
Experimental hemarthrosis in primates has not been reported 213 214 except for rare experiments involving humans. '
1 2
Interest in 'acute' hemarthrosis also stems from another naturally occurring problem, traumatic hemarthrosis. Traumatic hemarthrosis is common in sports injuries and is frequently associated with disruption of
IQ1 0 10 the anterior cruciate ligament. * y Experiments designed to look at the acute changes of hemarthrosis have been primarily concerned with the natural methods of removal of blood from joints and the effects of blood or its components on the synovial membrane and articular cartilage. 13,24,88,102,124,182,213,214,217 These projects utilized rabbits and concentrated on morphologic alterations. They have not included biochemical analyses, metabolic studies or morphometric analyses. These parameters are important in detecting subtle changes that may occur early in the pathogenesis of blood-induced arthropathy.
Although few species have been utilized in experiment al hemarthrosis studies, there is some indication of species variability. In the rabbit and dog multiple injections of blood over a prolonged time period were required to produce significant changes in the articular
119 91 990 cartilage.^*1 But in the horse, a single injection of autogenous blood into stifle joints produced 1 9 fibrillation in all animals within three months. Where primates fall in this broad spectrum of sensitivity is uncertain because experiments with humans have been Oio 91 A understandably limited, * and experiments with nonhuman primates have not been reported.
Interest in hemarthrosis of nonhuman primates at
Louisiana State University was initiated by the investigations of Roberts et al into the pathogenesis of naturally occurring calcium pyrophosphate dihydrate- deposition disease (CPPD-DD) in rhesus monkeys (Macaca mulatta) from the Delta Regional Primate Research Center in
Covington, LA. One observation made by them was that several of the affected individuals had prior joint trauma resulting in hemarthrotic episodes as evidenced by synovial membrane hemosiderosis and periarticular scar tissue.
These monkeys were housed in large outdoor breeding colonies and fighting among individuals was not uncommon.
One of the favored locations for bite wounds to occur was the femorotibial joints, CPPD-DD is primarily a disease of humans which still has an obscure pathogenesis. It is a crystal-related arthropathy with numerous disease
i f\ n associations. The associated diseases are a heterogenous group, indicating that a variety of conditions may result in increased or deranged metabolic activity of the chondrocytes, especially with respect to pyrophosphate metabolism.32,205 However, the strongest evidence for a significant association exists for primary 205 hyperparathyroidism and hemochromatosis. In both of these conditions, the mechanism may be interference with o 0 the metabolism of inorganic pyrophosphate (PPi). ^
Pyrophosphatase, a crucial enzyme required for the breakdown of PPi into soluble components, is strongly inhibited by divalent metal cations such as Fe++ and
Ca++,^5>66,159,165,268 These cations are elevated respectively in tissues from individuals with hemochromatosis and hyperparathyroidism, and are believed to inhibit the breakdown and allow the accumulation of endogenous PPi. Accumulated PPi may then precipitate into insoluble calcium pyrophosphate dihydrate crystals. Iron present in heme is in the divalent form and theoretically may act in a similar manner, to inhibit pyrophosphatase during a hemarthrotic episode.
Considering the possibility of trauma association in
CPPD-DD in rhesus monkeys, a hemarthrosis experiment was designed. The primary objective of the experiment was to determine the role of hemarthrosis in the pathogenesis of local metabolic disturbances leading to CPPD-DD. Theories regarding the pathogenesis of CPPD-DD could be tested for the first time in a species clo s e l y related to man in whic ono the disease is now known to occur naturally. The experiment should also fill a void in experimental information about hemarthrosis in primates, and aid evaluation of the 'early' degenerative changes that occur following a single hemarthrotic episode. To accomplish th latter, biochemical analyses, metabolic studies and morphometric analyses were added to the traditional morphologic evaluations. LITERATURE REVIEW
Anatomy, Physiology and Pathology of Synovial Joints
Joint Development
Skeletal articulations have traditionally been
classified on the basis of joint mobility, and articular
histology. The synovial joint is a diarthrodial (freely
movable) articulation which separates cartilage capped bony
surfaces by an articular cavity lined by a synovial
raembrane,^^ In primates and most quadripeds, major
synovial joints of the body include the scapulohumeral,
cubitus, radiocarpal, coxofemoral, femorotibial and
tibiotarsal joints.
The most important components of a synovial joint
include the articular cartilage, subchondral bone plate, articular capsule, synovial fluid and, in some cases, intra-articular discs. Joint development and the formation of these structures occur in three separate but interrelated events a) segmentation of the cartilagenous
limb with the formation of an articular surface at the end of each segment; b) joint cavity formation; c) and development of intra-articular structures.57,69,103,246,247
First appearances of segmentation in the human knee joint occur by stage 17 or 18 in human embryos.^ Concentric rings of flattened c e lls form at the ends of the cartilagenous primordium. This cap of flattened chondrocytes is separated from the adjacent bone by a zone
6 7 of undifferentiated mesenchymal tissue known as the interzone. Later, the interzone develops a loose central zone and two dense peripheral zones. The dense peripheral zone adjacent to the end of the bone anlage becomes a primitive perichondrium which later gives rise to the articular cartilage.^ ’ 103,246
Minute cavitations begin in the loose central area of 111 the interzone. Numerous small cavities coalesce to form the definitive joint cavity. Cavitation is preceded by vascular penetration; necrotic cells are apparently lysed and therefore not seen in the newly formed cavities; and hyaluronic acid can be demonstrated histochemical1y in the 7 111 cavitated areas. ' Motion of the embryonic joint is crucial for further development. In the absence of motion, degenerative changes will occur and the joint will become
CO fused by fibrous connective tissue. The primitive synovial lining is formed as the multiple cavitations coalesce. Soon after this, blood vessels penetrate the subintimal tissues, followed later by the development of articular fat pads and innervation.^^
As the epiphysis continues to grow, chondrocytes which originated from the interzone, continue to multiply to keep the expanding epiphysis covered by articular cartilage. At this point, the epiphysis is entirely cartilagenous and nourished by vascular canals. Sometime following establishment of the primary center of ossification in the 8 diaphysis, a secondary ossification center is established in the center of the epiphysis. Ossification expands in a centrifugal fashion until the cartilage anlage of the epiphysis is completely ossified. The epiphysis continues to enlarge with growth by minor growth plates at either end and by periosteal bone growth at the sides. Thus at the articular end of the epiphysis, there are two zones of 07 i on chondrocyte replication. * ^ One zone of replication maintains the articular cartilage over the expanding epiphysis and the other zone is for the enlargement of the epiphysis via endochondral ossification. This dual purpose of the epiphyseal cartilage endplate during development has led to the proposal in terminology for this structure as the 'articular-epiphyseal cartilage complex,.'^»^^*^^ At the end of skeletal growth, mitotic activity ceases in the articular-epiphyseal cartilage complex and a thin horizontal plate of bone forms beneath the articular cartilage to become the supportive subchondral bone plate.
Mitotic activity in adult articular cartilage is not recognized again except in degenerative and reparative situations.
Gross Anatomy
The gross appearance of normal articular cartilage is smooth and glossy. Color varies slightly with age; it is blue-white in the very young, white in young adults, and
o n q 9 A S yellows slightly with age. * Close examination will 9 reveal a few slight surface irregularities. Articular cartilage lacks lymphatics, blood vessels and nerves; and therefore presents a homogenous appearance throughout.
Thickness of the articular plate varies from 1-7 mm and will generally be thicker over major gliding or' weight bearing regions.^4^ The thickness of articular cartilage covering the femoral condyle of rabbits is slightly less than 0.5 mm.^ The thickness of femoral articular cartilage in rhesus monkeys has not been reported, but in humans is
2-4 mm thick.74
Although the above gross descriptions are accurate for the femoral side of the femorotibial joint, the tibial articular surface is quite different. The femoral condyles articulate with counterpart fibrocartilagenous menisci.
The menisci are crescentic structures positioned on the tibial plateau and held in position by peripheral attachments to the fibrous capsule and ligamentous attachments to the tibia and its tuberosities. 10*76,116
Although menisci are predominantly composed of fibrocartilage, they also contain elastic fibers which give them resiliency for functioning as an intra-articular shock absorber. The articular cartilage of the tibia is mostly covered by menisci and therefore is not a gliding articular surface. In rhesus monkeys, the tibial surface is composed of thin, smooth hyaline cartilage peripherally, but contains a central fibrocartilagenous region comparable to 10 a fibrous articular surface.This fibrocartilagenous area is not an articulating or weight-bearing surface and roughly corresponds to the open region of the crescentic menisci.
The articular capsule encloses the entire joint cavity and is composed of two distinct connective tissue layers: a thick tough outer layer, the fibrous capsule, and a thin 203 delicate.inner layer, the synovial membrane. The fibrous capsule is formed by dense white fibrous connective tissue and is attached to the periosteum of articulating bones. It contains many blood and lymphatic vessels and nerves. The synovial membrane is a pink, glistening membrane lining the inner articular capsule, and intra- articular ligaments and tendons. It is generally flat but may contain barely visible gentle folds, especially around menisci and recessed portions of the joint. It is an extremely thin membrane and therefore accumulations of lipid beneath the membrane are clearly visible. Large accumulations of lipid are specialized intra-articular structures called fat pads. Fat pads are covered by a flattened layer of synovial cells and are believed to 203 function as shock absorbers or cushions. Patellar fat pads are prominent in femorotibial joints and are located just proximal to the patella.
Microscopic Anatomy of Cartilage
Articular cartilage consists of cellular 11
(chondrocytes) and extracellular components (ground substance or matrix). The extracellular matrix consists of 2 57 fibrillar and nonfibrillar components. The fibrillar matrix is predominantly type II collagen, but there also are a few fine unbanded fibrils present. The collagen
o tr 7 fibers in cartilage range from 5-200 nm in diameter.
The collagen fibers in cartilage cannot be seen with routine light microscopic examination because they are not stained well, presumably because their cationic staining sites are masked by the g1ycosaminog1 yeans.^ However, they are visible under polarized light. Middle-aged human femoral head articular cartilage is approximately 55-65% 277 collagen by dry weight. ' ^
The nonfibrillar component of the matrix is composed of water (76%), proteoglycan (18% dry weight), and necrotic 27 2 cellular debris. Ultrastructurally, proteoglycan particles apppear in the matrix as 30-70 nm electron-dense particles, sometimes called matrix granules.^ Although proteoglycan particles cannot be seen by light microscopy, sections of articular cartilage stained with hematoxylin and eosin show cartilage to have an abundant, basophilic matrix. The basophilia is due to the metachromatic stain reaction with proteoglycans in the matrix. When sections are stained with safranin 0, areas rich in proteoglycan will stain rosy red/^^^13 Since proteoglycan content is greatest in the radial and transitional zones, and least in 12
the tangenital zone, basophilia and safranin 0 affinity
corresponds accordingly. Matrix zones can be seen by light
microscopy due to local differences in the staining
intensity. These differences are not necessarily due to
differences in proteoglycan content in these regions, but
are believed related to the presence of other chemical
components in these regions, such as non-collagenous
protein, which may compete with the cationic dye for 9 £ Q binding sites. This theory is further supported by
biochemical analysis of the various matrix zones, which
shows a fairly homogenous distribution of g1ycosaminog1 yean 9 f* 9 in cartilage. There are three cartilage matrix zones:
a) the pericellular matrix is a lake of fine textured
material immediately surrounding the chondrocyte and is
composed of proteoglycan particles, filaments and fibrils;
b) the territorial matrix is an increased zone of
basophilia immediately around individual lacunae or
isogenous groups; c) the inter-territorial matrix is the
n r 7 remaining matrix between chondrocytes.
U1trastructurally there is no distinction between the
territorial and inter-territorial matrices. The lacunae can thus be defined as the limit of encroachment of the collagenous matrix around the chondrocyte and contains both o c 7 the pericellular matrix and the chondrocyte.
Articular cartilage has been traditionally divided into four horizontal zones at the light microscopic l e v e l . ^ Zone I, or the tangential zone, is adjacent to the joint space and is composed of oval or elongated chondrocytes. It contains numerous tightly packed fine collagen fibers which are arranged tangentially to the articular surface. This tightly packed arrangement creates an extremely tough impermeable surface layer, occasionally referred to as the 'armor-plate layer1. Zone II
(transitional zone) is composed of individual round chondrocytes and collagen fibers that are in a transition between being arranged perpendicular or tangential to the articular surface. Collagen fibers anchored in the deepest zone of the articular cartilage extend upward perpendicular to the articular surface and then arch in the tangential zone. It is this arching, which gives the zone II collagen fibers their transitional position and appearance. In zone
III (radial zone) the collagen fibers are arranged perpendicular to the articular surface and chondrocytes are arranged in isogenous groups or radial columns. Collagen fibers are thickest in the radial zone. Zone IV (calcified zone) is separated from zone III by a thin wavy basophilic line termed the tidemark. The matrix of the calcified zone is impregnated by calcium salt crystals. The calcified zone is hypocellular and most chondrocytes appear degenerate or necrotic. The calcified zone interdigitates with the subchondral bone plate and thus anchors the articular cartilage firmly to the bone. The bone-cartilage interface 14
is sometimes referred to as the 'cement line', and the
nature of attachment at the cement line varies between
species. In adult humans, squirrel monkeys and dogs it is
formed by an irregularly arranged meshwork of collagen
I *[ O fibers mixed with mineral crystals. However, in growing
children, rodents and rabbits it lacks the collagen
component.
Chondrocyte ultrastructure has been studied in detail
by Ghadially71’74 and others.185)221’239"241,275 The
uniqueness of the chondrocyte is due to its relationship
with the extracellular matrix, for ultrastructurally it is
similar to other connective tissue cells. The nucleus
usually presents a smooth contour with only slight surface
irregularities. In older animals, deeper irregularities and even pseudolobulation of the nucleus can be present.
There is a double-membrane nuclear envelope with nuclear i a/, pores and a nuclear fibrous lamina. Nucleoli are small and therefore rarely seen. Chromatin is usually peripherally located or in small clumps elsewhere in the nucleus.
The cytoplasm of chondrocytes contains the usual variety of organelles and is contained by a double membrane. Centrioles are rarely seen and even more rarely have been shown to form the basal body of a single cilium. Mitochondria are the same as in other cells and slightly larger and more abundant in the transitional and 15 radial zones. Mitochondria are occasionally associated with lipid droplets. The endoplasmic reticulum, Golgi apparatus, secretory vesicles and lysosomes are also present in greater numbers in the transitional and radial zones. Microtubules can best be demonstrated by using' glutaraldehyde fixation only, without osmium tetroxide, at room temperature. Bundles of intracytoplasmic filaments, presumably vimentin, are commonly seen in radial zone chondrocytes of healthy animals..^ They are seen less frequently in transitional zone chondrocytes and only occasionally in tangential zone chondrocytes. Large accumulations of filaments are usually located in the perinuclear position, and are closely packed in parallel arrays.Intracytoplasmic lipid is usually seen as one / Q or two droplets in 25-50% of the chondrocytes. Lipid droplets are seen more frequently in transitional and radial zone chondrocytes. Intracytoplasmic lipid is apparently derived from the joint lumen for corn oil experimentally injected into the joint of animals led to increased number of lipid droplets within oc i A 7 9 R 7 chondrocytes. ’ * Glycogen in chondrocytes occurs only in the monoparticulate form as 15^30 nm electron-dense particles. Glycogen is most abundant in the radial zone chondrocytes and is often present in large accumulations when large amounts of cytoplasmic filaments are also present. Chondrocytes are bound by a tri-laminar membrane 16 and have irregular cytoplasmic borders in the radial zone and less so in the transitional and tangential zones.^
Convoluted chondrocytes usually also have cell processes which may extend to the outer border of the pericellular matrix.
In summary, there is u1trastructural evidence of zonal differences in the cytoplasmic components of chondrocytes.
There is a progressive increase in the content of endoplasmic reticulum, Golgi apparatus, mitochondria- and lysosomes proceding downward through the various cartilage
9 c zones to the calcified zone. The calcified chondrocytes contain few organelles and increased numbers of clear cytoplasmic vacuoles that are interpreted as a degenerative change.
The surface of articular cartilage at the light microscopic level appears relatively smooth and slightly convex to conform to the shape of the underlying bone.
Several investigators have commented on the many small surface undulations and furrows that are present when viewed by scanning electron microscopy. 16,50,153,200,275
However, these studies utilized cartilage detached from bone which curls and wrinkles during processing. It has been shown in several species that when cartilage is processed attached to bone, the surface is markedly smoother with only minimal irregularities, usually less than 0,03 urn deep.^®*^®*^ >59,67,80,82,83 transmission 17
electron microscopy (EM) a thin layer (0.03-.1 um) of
particulate and filamentous material can be seen on the
cartilage surface. Ghadially referred to this material as
the 'surface coat’ and suggests that it is partially formed
by matrical lipidic debris discharged from the articular D ft cartilage. Scanning EM also reveals small humps and pits
on the surface. These are drying artefacts; the pits
o n represent collapse of the matrix over a lacunae.
Synovial fluid on articular cartilage and synovium appears as variably sized round droplets, sheets, strands, or
pools. 273
Microscopic Anatomy of Synovium
The synovial membrane is composed of a delicate inner
cellular layer, usually one to four cell layers thick, termed the synovial intima; and a supportive and vascular 7 Q layer of connective tissue called the synovial subintima.
The microscopic appearance of the membrane has been described by several authors in different species.15’31’45’*8’79’87’131’210’218’236’284 The synoviocytes are loosely set in the intimal matrix and do not form cellular attachments. Synoviocyte shape varies from round to polygonal to elongated. Elongated synoviocytes usually line intra-articular ligaments and tendons. The membrane is not a continuous layer and gaps will be present where the matrix is apparently exposed to the joint space. By staining light microscopic sections 18 with methyl-green pyronin to demonstrate the RNA of ribosomes on endoplasmic reticulum, two, possibly three, cell populations can be distinguished. Using this method,
3-4% of synoviocytes were markedly pyroninophi1ic, 19-41% were slightly pyroninophilie, and 54-81% were not 79 pyroninophilic. This suggests populations of secretory, non-secretory and intermediate synoviocytes exist.
The synovial subintima is classified according to the predominant tissue type composing it. The three major types are adipose, areolar and fibrous. Combination types are also designated, such as fibro-areolar or areolar- adipose. Other components of the subintima include fibroblasts, macrophages and lipocytes. Mast cells have been seen in the subintima of cat, dog, sheep, monkey and man but not guinea pig or rabbit.238
Transmission electron microscopy reveals that synoviocytes are loosely arranged in a matrix composed of banded collagen fibrils, unbanded fibrils and electron- dense amorphous material which may contain a few filaments 7 9 and particles. y Gaps between cells and direct exposure of the intimal matrix to the joint space is confirmed by TEM.
Several studies also confirm the absence of a true basememt membrane.4 8 *8 7 '9 8 -2 1 0 *2 1 8 *2 3 3 *284
There is disagreement among authorities regarding the different types of synoviocytes present. Ghadially states there are two major synoviocyte types (types A and B) and an intermediate type (AB).^ The percentage of each type present at a given time varies greatly and according to
Ghadially this is because the types merely represent morphologic differences of the same basic cell line at different functional times. Using Ghadially's nomenclature, type A cells contain a prominent Golgi complex, numerous vesicles and vacuoles, and scant amounts of rough endoplasmic reticulum (RER). The type B cells contain abundant amounts of RER but scant numbers of Golgi complexes, vesicles , and vacuoles. Type AB cells contain abundant amounts of both RER and Golgi complexes. Although older literature simplistical1y refer to type A cells as
'phagocytic' and type B cells as 'secretory', Ghadially contends that both types can have secretory and phagocytic functions.^ Ghadially states that type AB or B cells usually predominate, but shifting of cell populations is apparently possible.^ This has been shown experimentally in rabbits where increased numbers of type A cells were
n 1 7 found following acute hemarthrosis.
Graabaek recently challenged Ghadially on two major points: 1) he claimed that only two synoviocyte types exist, type A and S, 2) and that these types were distinct cell types and not interchangeable.9?t98 Using serial sections of rat synovial membrane Graabaek showed that synoviocytes that appeared to be type AB (using Ghadially's nomenclature) in one plane appeared to be type B in later Q ft serial planes. Graabaek proposes terminology and function for these two cell types: a) type A or absorptive macrophagic cells and b) type S or secretory cells. The type S cells are usually positioned deeper in the intima than the type A cells which generally line the joint space.
Other major morphologic differences allow the identification of type, even from partial cell profiles.
Type A cells have : a heterochromatin rich nucleus, slender surface projections, absence of caveolae, poorly developed RER with narrow cisternae, wide pale mitochondria, infrequent Golgi complexes, no dense granules, numerous diffusely distributed vacuoles, many lysosomes, and no cilium. In contrast, type S cells have: a euchromatin rich nucleus, broad surface projections, numerous caveolae, extensively developed RER with distended cisternae, slender dense mitochondria, frequent and large
Golgi complexes, occasional dense granules, a few vacuoles only in the cell processes, few lysosomes, and often a single cilium. Graabaek's work is convincing in the rat and appears thourough, but whether his concepts will be true for other species remains to be evaluated. Although most of Ghadially's work has been done on human and rabbit synovial membrane, he has also published some work with 21ft rats. 10 Nonetheless, Ghadially still maintains that type
AB cells are a universal component of the synovium in most species.79 21
The subintima ultrastructurally appears as would be expected from the light microscopic studies and contains
varying amounts of lipid and collagen depending on the
synovial membrane type. Capillaries are numerous in the
subintima and rarely may be present in the intima.
Capillaries in fibrous synovium are of the continuous type, but capillaries in adipose and areolar synovium are 7q o 88 fenestrated on the side facing the joint space.
There have been few scanning EM studies on the synovial membrane compared to the number of transmission EM studies. 1°1>200,238,282,285 Ghadially does not even include a scanning EM photograph in his recent book and claims that the method is fraught with technical problems for synovial membranes.^ The main problems are that the synovial fluid adheres to and obscures the cells; and that synoviocytes are loosely and delicately attached to their matrix which precludes attempts to rinse away the synovial fluid prior to fixation. Examples of these problems are 1 01 9 R 2 9 R S evident in some studies, ’ ' but useful topographic 900 988 information is present in other studies, ’ The topography corresponds somewhat to the subintimal tissue and synovium type. Fibrous synovium is usually lined by flat synoviocytes; adipose synovium has a cobble stone appearance created by the underlying adipocytes; and areolar synovium is lined by round synoviocytes. Villi are long finger like projections lined by synoviocytes and are 22 frequently present in areas where the synovium reflects upon itself or around intra-articular structures.
Microscopic Anatomy of Menisci
The meniscus is composed of fibrocartilage and at the light microscopic level, three layers can be seen when cross sectioned.The two superficial zones (dorsal and ventral) closely resemble cartilage in their appearance while the middle zone resembles coarse collagenous connective tissue. In the superficial zones, chondrocytes are present in lacunae and are surrounded by a glassy homogenous matrix. The middle zone is composed of large collagen fibers (also cut in cross section) and thin fibroblast-like cells. The surface is smooth to slightly undulating. Calcification is not uncommon in joints from older humans with osteoarthritis or rheumatoid arthritis, 7 6 but is rare in young humans.
Transmission EM studies of menisci confirm the tissue is fibrocartilage as chondrocytes, fibroblasts and intermediate type cells can be identified. ^
Chondrocytes are surrounded by a more abundant pericellular matrix of filaments and fibrils than are hyaline cartilage chondrocytes. The territorial matrix contains coarse collagen fibrils and numerous proteoglycan particles. The surface of menisci as viewed by scanning EM is quite similar to articular cartilage and displays the same type of fixation artifacts, such as ridges, furrows, humps and Physiology of Articular Cartilage
Like other connective tissue cells in the body, much of the metabolic efforts of chondrocytes are directed towards maintenance of the surrounding matrix.
Chondrocytes are unique in that they accomplish this in an avascular environment. Although several enzyme systems
(the glycolytic pathway, the pentose and tricarboxylic acid cycles) have been demonstrated in cartilage, anaerobic metabolism predominates,^^’ There is even evidence suggesting chondrocytes need an anaerobic environment for differentiation, and that synthetic activity
17ft 1 ft ft increases in low oxygen tensions. * The metabolic activity of chondrocytes has been considered extremely low, but this is not the case if one considers the low cellularity of cartilage relative to other connective tissue types. In the avascular environment of cartilage, nourishment is received through constant bathing in synovial fluid. The calcified cartilage zone of adult cartilage inhibits the movement of nourishment from the marrow side. However in growing animals, the epiphyseal cartilage has not closed and additional nourishment can come from the bone marrow vasculature and vascular cartilage canals.
The major components of the matrix are collagen fibers and proteoglycans. The collagen fibers in the tangential 24
zone are tightly packed, allowing the diffusion of small
molecules (ions and glucose) while virtually excluding the
diffusion of larger molecules (proteins and hyaluronic
acid). Collagen content is greatest at the surface of
articular cartilage while proteoglycan content is greatest
in the deeper zones of articular cartilage. Articular
cartilage is composed of type II collagen and the building
block components are produced by chondrocytes and released
to the matix. Many review articles on collagen
biosynthesis are available and a brief description of the
process in chondrocytes f o 1 1 ow s . ^ ^ ’ ^ ^Initially,
pro-alpha-chains are formed on the RER of the chondrocytes.
These chains are rich in lysine and proline and are
modified within the cisternae of the RER. Proline and
lysine are hydroxylated by prolyl and lysyl hydroxylases
with Fe++, ascorbate, and alpha-ketoglutarate as cofactors.
This is followed by glycosylation. Three modified pro-
alpha-chains form a triple helix called procollagen.
Procollagen is moved through transitional endoplasmic
reticulum to Golgi complexes for packaging in secretory
vesicles. Secretory vesicles fuse with the chondrocyte
cell membrane and procollagen is released outside the cell.
Extracellular procollagen peptidases cleave the N and C
terminals from procollagen to form tropocollagen.
Tropocollagen units assemble to form a collagen fibril which are covalently cross-linked by the action of lysyl- 25 oxidase with copper as a cofactor.
The other major matrix component of cartilage is proteoglycans. Numerous proteoglycan units aggregate onto hyaluronic acid to form extremely large proteoglycan aggregate molecules, 108-110,123 three-dimensional configuration of the aggregate has been compared to a test- tube brush; the hyaluronic acid is comparable to the wire core of the aggregate and numerous proteoglycan molecules radiate out from the core similar to the bristles on the
*) brush. This large molecule becomes entwined between the collagen fibers. Proteoglycans attract water and small ions into cartilage resulting in tremendous swelling pressure. The swollen proteoglycan molecules are firmly entrapped in the collagen mesh and the net result gives
9 cartilage its resilient characteristic. The sulfated glycosaminoglycans (GAG) of cartilage are chondroitin sulfates 4 and 6 , and keratan sulfate. These are repeating disaccharide units covalently linked to a core 1 1 O 1 9 ^ protein. ■LO,A ‘s Keratan sulfate is preferentially positioned closer to hyaluronate. The synthesis of proteoglycan by chondrocytes is similar to collagen in that the basic building blocks (GAG’s in this case) are produced within the cell and then packaged for secretion. Formation of the final functional unit occurs outside the cell i.e. proteoglycan aggregation. Proteoglycan synthesis begins when the core proteins are synthesized by RER. Link region 26
sugars are added to the core protein in the cisternae of the RER. The modified core protein passes through transitional ER to Golgi complexes where elongation of polysaccharide side chains and sulphation occurs. The completed proteoglycan molecule is packaged in secretory
vacuoles which migrate to the cell surface, combine with the cell membrane and discharge their contents. The proteoglycans then aggregate onto hyaluronic acid and become wrapped around collagen fibers to form an immovable proteoglycan-collagen-gel complex.
Certain hormones can effect chondrocyte metabolism, especially in growing individuals but also somewhat in adults. Hormones which promote synthesis in chondrocytes include growth hormone, thyroxine and insulin; while estrogen and cortisone generally inhibit chondrocyte synthesis.256
As noted earlier, chondrocytes are metabolical ly active in replacing their matrix. 143*266,267 glycosaminog1 yeans are apparently turned over at a more rapid rate than the collagen component of the matrix. It is estimated that in human articular cartilage, that one- half of the matrix glycosaminoglycans are replaced every year, while one-half of the matrix collagen would be ft 7 replaced only every ten years. All of the enzymes needed to degrade the cartilage matrix have been identified in articular cartilage except hyaluronidase which is 27
cc OOP 9 fi S present in the synovial membrane. Enzymes are
released into the matrix and partially degrade the
macromolecules into diffusable fragments. Fragments are
taken up by endocytosis and further digested in
phagolysomes. The important enzymes involved are: the
cathepsins (B1 and D) which can degrade collagen and 228 proteoglycan; ^ 0 other metalloproteases capable of
2 2 Q degrading cartilage proteoglycan; 7 arylsulfatase which is capable of initiating proteoglycan degradation; ° acid and alkaline phosphatases which are probably involved in Q Q degradation of the phosphate moieties of proteoglycans; ’ and lysozyme which can degrade carbohydrates by hydrolysis.1^0,129 Neither co1lagenase^ or 2 2 hyaluronidase have been found in normal articular cartilage.
Physiology of Synovium and Synovia
The synovial membrane has two major functions; the removal of unwanted material from the joint space by phagocytic activity, and the production of synovial fluid.
As discussed previously, the two types of synoviocytes either perform these functions separately (i.e. specialized cell types as proposed by Graabaek) or jointly (as proposed by Ghadially). The synovium is nourished by the vascular- rich subintima. Capillaries in adipose and areolar synovium are fenestrated on the side facing the joint space.79,258 This fact, coupled with the fact that 28 synoviocytes are loosely arranged in the intimal matrix with few cellular attachments, permits the free diffusion of blood plasma across the intima and into the joint space.To this transudate of blood plasma are added hyaluronic acid and other proteins which collectively form
1 7 7 the synovial fluid. Most of the added components are contributed by type S cells according to Graabaek;^’^® or 7 Q by any of the three cell types recognized by Ghadially.
The absorptive function of the synovium is necessary to remove degradation products and to prevent the excessive accumulation of synovia which is continually being produced. Most synovial fluid is removed via micropinocytotic uptake by synoviocytes.79,97,98 Several experiments have confirmed this by the intra-articular injection of various substances, including: iron 1 >i 177 A 2 dextran, ferritin, thorium dioxide, colloidal carbon,^ and colloidal gold.^® This material is then found in phagosomes and phagolysomes of the absorptive synoviocyte. Graabaek believes phagocytic activity is the function of his type A synoviocyte exclusively.^’^®
The two major functions of synovial fluid (synovia) are to carry nourishment to the articular cartilage and to £ Q lubricate the articular surfaces. Nourishment is accomplished as synovia is distributed across the articular surfaces and components are absorbed. The lubricating qualities of synovia are due to its mucin content. Mucin 29
is a combination of hyaluronate and protein elements. The
low coefficient of friction in joints is due to a
combination of lubricating mechanisms; fluid lubrication and a specialized boundary lubrication.In fluid
lubrication, the moving surfaces are separated by a continuous film of lubricant. In boundary lubrication, each weight bearing surface is coated or impregnated with a thin layer of molecules which slide on the opposing surfaces more readily than they would shear. Articular cartilage is lubricated at low weight-bearing loads by boundary lubrication (glycoprotein coating) and at high
loads by fluid lubrication of the squeeze film type.^^
Lubrication of the soft tissues is also important in joint motion. This is also accomplished by the highly viscous synovial fluid which adheres to and keeps synovial membranes from sticking to one another and to cartilage surfaces.
General Pathology of Synovial Joints
A general classification of joint disease by etiology contains five major groups: infectious disease, possible infectious disease, degenerative disease, traumatic
o n fi disease, and metabolic disease. However, when considering the general pathologic reactions of the joint to injury, two broad categories can be useful: 187 inflammatory and noninflammatory joint disease.
Inflammatory joint disease is characterized by inflammatory 30 changes in the synovium and synovia, with systemic signs of illness such as fever, malaise, leukocytosis, anorexia, and hyperfibrinogenemia. This is in contrast to noninflammatory joint disease which is characterized by absence of synovium inflammation, essentially normal 187 synovia, and no systemic signs of illness. Although there can be considerable overlap in the two categories
(for example osteoarthritis can be accompanied by periodic inflammatory flares secondary to release of degraded cartilage products), consideration of these separately will follow,
Inflammatory Joint Disease
Infectious and rheumatoid arthritis are two of the most extensively studied inflammatory joint conditions.
Enzymes capable of destroying the cartilage matrix and hence the integrity of the articular cartilage itself are major factors in the pathogenesis of these diseases. Both conditions involve a hyperplastic synovial membrane reaction which results in increased intiraal vascularization and a resultant influx of inflammatory cells and their enzymes into the joint. The hyperplastic synovium itself is composed of increased numbers of synoviocytes which also produce various catabolic enzymes. In rheumatoid arthritis, all the enzymes necessary for the degradation of the matrix are present in the synovium or the synovia.^’ ' -^0, 171,187,267 enzymes released from 31 influxing neutrophils also play a role in rheumatoid 1oo 9 9 9 arthritis cartilage degradation, ’ but not to the same degree as in infectious arthritis.46,47,49,120,248 These degradative enzymes destroy the architectural integrity of the cartilage by first depleting the proteoglycan component of the matrix. Decreased resiliency is a direct result of proteoglycan loss and the cartilage eventually becomes soft
(chondromalacia) and incapable of normal weight bearing.
This leads to further destruction by mechanical factors, because the chondromalacic areas are extremely susceptible to shear forces. Surface changes which can be identified grossly are erosions (shallow cartilage defects), ulcers
(deep cartilage defects) and eburnation (total loss of articular cartilage with polishing of subchondral bone due to friction). At the edges of the joint, the hyperplastic synovial membrane gradually encroaches over the articular surface. This phenomenon is termed pannus and when it occurs, the articular cartilage beneath the pannus rapidly is destroyed by enzymatic methods. In rheumatoid arthritis, the chronic inflammatory reaction may be sustained by an immune response in the synovial membrane to I £ Q the products of matrix degradation. Other mediators of the inflammatory reaction in both infectious and rheumatoid 167 911 arthritis include prostaglandins, ’ interleukin- 19R 980 ino 1 , • connective tissue activation peptide, and I QQ plasminogen activator. Prostaglandins have been shown 32
to suppress proteoglycan synthesis in rabbit chondrocyte 1 *37 cell cultures. The erosive effects of pannus formation
may be enhanced by the inhibition of anti-vascular factors
present in cartilage.
The microscopic appearances of inflammatory joint
disease varies somewhat with the inciting cause and the
stage of the inflammatory reaction. In infectious arthritis, there is a marked fibrinopurulent exudate in the synovium. In a few days, the intima and subintima may be replaced by a bed of granualtion tissue, heavily infiltrated by neutrophils. Multifocal necrosis and sloughing of synoviocytes is common. In infectious arthritis the inflammatory reaction is acute and the synovial membrane usually does not have time to react by hyperplasia, although hyperplasia may be evident as the reaction subsides and the lesion is resolving.
In marked contrast, the inflammatory reaction of rheumatoid arthritis is characterized by a chronic hyperplastic and exudative synovitis.^• ^ Synoviocyte hypertrophy, and synovial villi hypertrophy and hyperplasia are common. Focal areas of necrosis and synoviocyte sloughing are replaced by thick layers of fibrinoid material. The inflammatory cell infiltrate in the synovium is predominated by lymphocytes and plasma cells, and occasional lymphocytic follicle formation is present.
Pannus and erosion of articular cartilage is common at the 33 joint periphery. Evidence of chronic multifocal hemorrhage
1 7 0 I 7 C is frequently present, ' 1'J and hemosiderin laden macrophages accumulate in the subintima.
The fine structure of synovial cells in rheumatoid 70 ooq arthritis deserves special mention. ’ The synovial intima is thickened by increased numbers of tightly packed synoviocytes. Many of these cells are larger than normal
(hypertrophic) and some have fused to form multinucleated giant cells. Hypertrophy appears to be related cytoplasmical ly, to a marked increase in RER. Therefore the predominate cell is compatible with the type A or AB 7 ft cell of Ghadially. There is marked thickening of the nuclear fibrous lamina in many synoviocytes,'*®^ There is also an apparent paucity of Golgi complexes and secretory vesicles, which is interpreted as an indication of decreased hyaluronic acid production and may partially explain the low levels of hyaluronic acid found in the synovia of rheumatoid patients. Lysosomes are increased in numbers as well as' accumulations of intracytoplasmic filaments. Increases in intracytoplasmic filaments are found in endothelial cells also. The deposits of fibrinoid, as seen by light microscopy, are composed of banded filaments (20-35 nm periodicity) compatible with fibrin.
Noninflammatory Joint Disease
Categories to consider in noninflammatory joint 34 disease include osteoarthritis (osteoarthrosis, degenerative joint disease), deve1 opraenta 1 arthropathies, arthropathies secondary to inborn errors of metabolism, dietary arthropathies and neoplastic arthropathies. An extensive amount of literature exists on osteoarthritis, due to its major significance in humans. Therefore, the following discussion will be limited to osteoarthritis as a general example of noninflammatory joint disease.
Osteoarthritis has generally been divided by etiology.
Secondary osteoarthritis is that which develops following a known joint insult such as trauma, conformational defects, or resolving inflammation. Primary osteoarthritis is idiopathic and usually appears later in life. Clinically, osteoarthritis is a slowly progressive disorder, primarily affecting the weight bearing joints, and associated with pain and limited joint motion. Pathologically, osteoarthritis is characterized by focal erosions and cartilage destruction, subchondral sclerosis and cyst formation, and large osteophytes at the margins of the joint. 140»3,41
At the light microscope level, the initial changes are seen at the articular surface. Fibrillation appears to be the result of disruption of collagen fibers in the tangential zone and appears as horizontal splitting and flaking at the surface. There may be a moderate decrease in metachromatic staining at this time, indicative of slight 35 proteoglycan depletion. With progression, vertical clefts begin to form, starting at the surface and elongating downward into deeper zones. Vertical cleft formation is compatible with the arrangement of collagen fibers which are vertical throughout most of the radial and transitional zones. The tidemark line may be crossed by blood vessel ingrowth from the underlying bone. Chondrocyte cloning is common, especially near fibrillated areas and along clefts.
Clones are the result of chondrocyte proliferation during repair attempts. In moderate to severe osteoarthritis the matrix is severely depleted of proteoglycans and metachromatic staining (such as safranin-O) is poor.
Further cartilage destruction may lead to denudation and the underlying bone becomes sclerotic and eburnated.
The above microscopic description is considered the
T I 1 classic picture of progressive osteoarthritis, however
Sachs et a]_95,226 have recently described three early histopathologic cartilage types in human osteoarthritis
(types A, B, and C). Type A is composed of evenly distributed chondrocytes with a deficient metachromatic stain about the lacunae. Type B tissue is characterized by clones of small spheroidal cells with intense periclonal matrix metachromasia. Type C cartilage is a mixture of small stellate chondrocytes and round chondrocytes, evenly distributed and with irregular matrix-staining characteristics. 36
U1trastructura1 changes in osteoarthritic cartilage as noted by Ghadially include: swollen mitochondria, dilated endoplasmic reticulum, several Golgi complexes and secretory vacuoles, increased numbers of lysosomes, massive accumulation of intracytoplasmic filaments, increased matrical lipidic debris, increased pericellular matrix, and 7 3 occasional giant collagen fibers. These changes are especially prominent in chondrocytes near fibrillated or clefted cartilage whereas other areas would appear essentially normal.
Although noninflammatory joint disease lacks the characteristic synovial membrane reactions typical of inflammatory joint disease, articular cartilage destruction is still primarily mediated by enzymatic mechanisms. Once the cartilage matrix is disrupted and the armor plate layer is destroyed, synovial fluid and its components can readily penetrate the deeper cartilage zones. The release of cartilage breakdown products into the joint space, stimulates a local, variable inflammatory reaction in the synovial membrane. This produces a cycle of continual cartilage degradation and simmering inflammation in the synovium, which slowly progresses over months and years.
Virtually all of the enzymes responsible for cartilage destruction in inflammatory arthritis also play a role in the destruction of osteoarthritic cartilage. The distinguishing differences being the initiating factors and 37 the concentrations of the involved enzymes which result in different rates of degradation. In osteoarthritis in general, the enzyme concentrations are lower and factors initiating the original surface destruction are poorly understood. Some of the major enzymes involved in osteoarthritic cartilage destruction include the following: neutral proteases are capable of proteoglycan degradation at a neutral pH similar to the pH of the interterritorial matrix;17,136,151,228,230 aryisuifatase B is a lysosomal no/ enzyme capable of degrading chondroitin sulfate;'*'3 cathepsins B and D are lysosomal acid proteases capable of degrading proteoglycan intracellularly or in the
i o n n n o n o £ pericellular matrix; 3 1 ,WD and collagenase, which is only found in osteoarthritic cartilage, is capable of collagen degradation.^ * * ^78
Recent research in osteoarthritis has revealed the importance of cartilage-synovium interaction in modulation of the degenerative process.^^’^ ^ Cartilage matrix degradation products can stimulate the macrophage-like type
A synoviocytes to produce a degradation messenger called
'catabolin*. Catabolin can stimulate other synovial cells
(called dendritic or stellate cells) and chondrocytes to release degradative substances such as proteases, I QC 2 SA collagenase and prostaglandin E2. * Catabolin may be chemically identical to interleukin-1.
The most striking and consistent biochemical change in 38 osteoarthritic cartilage is proteoglycan depletion.^*23, 142,161,260 Water content is usually increased in osteoarthritic cartilage, 1^,149 This has been correllated ultrastructurally with collagen £ disorganization in the perilacunar region, which possibly resulted from mechanical disruption by the imbibed water.
The collagen content of osteoarthritic collagen is generally not different from that of normal cartilage.^^^
There is an apparent shift from type II to type I collagen
Q ft 1 ftO content; ’ however, this probably occurs only in advanced stages of osteoarthritis when fibrocartilage is formed as a reparative response to the disease.
The metabolic activity of osteoarthritic articular cartilage is generally increased, both in regards to proteoglycan synthesis^^,‘^^ ,^ ^ ,^ ^ and collagen synthesis.63,64,132,144,180,202 However, one study has shown a decrease in collagen synthesis in degenerate foci of osteoarthritic dog cartilage.’*-'^ This was apparently due to decreased cellularity within the degenerate foci that were selected for the analysis. 39
Hemarthrosis
Acute Hemarthrosis
Acute hemarthrosis can occur naturally due to 219 trauma. The synovial membrane is well vascularized and
if torn blood can rapidly fill the joint. Acute
hemarthrosis may also result from intra-articular fractures
and is then usually accompanied by lipids. This has been
termed ' lipohemarthrosis' by Ghadial 1 Another cause
of acute hemarthrosis is in patients receiving 10 0 1 ? O 1 / £ heparin, ’ and during dialysis treatment. This has
rarely resulted in spontaneous hemorrhage into multiple
joints. Acute hemarthrosis has also been reported with
2 7 fi sodium warfarin therapy and may occur with warfarin
toxicity. Interest in acute hemarthrosis experimentally
has been to determine the routes of blood removal from
joints•*■^,''•®^,^ ^ and to study the initial lesions towards 88 17i 217 the progression of chronic hemarthrosis. ’ ’ 10/ Key was one of the earliest investigators to study acute hemarthrosis. Using adult rabbits given a single injection of homologous blood and following the effect for
12 days he noted: congestion and leukocytic infiltration after one day which subsided after four days; synoviocyte hypertrophy which peaked at day five and regressed by day
1 2 ; and infiltration of the subintima by macrophages which peaked at day five and gradually subsided. Key concluded that blood could be removed from the joint by four routes: 40
by passing intact through the intima and then being
phagocytized by macrophages in the subintima; by being
phagocytized by macrophages in the joint lumen which then
migrate to the subintima; by being incorporated into the
intima as small clots; and by leaving through adjacent
muscular sheaths.
All of the potential escape routes of erythrocytes
have not been verified. Rodnan^^ designed an experiment
to clarify some of the possibilities by injecting
radiolabeled erythrocytes into rabbit knees. The escape of whole erythrocytes through the synovium and their return to
the general circulation was not confirmed, for he found no
radioactivity in the peripheral blood. The experiment did
confirm the rapid removal of intra-articular blood, for
approximately one-half of the radioactivity was removed
from the joint within 48 hours. However, 22% of the original radioactivity remained for at least a month
following the injections. Rodnan*s experiment^^ and 1 QO others also verified that joint motion enhances the
removal of blood from the joint. 917 Roy and Ghadially provided the first evidence of erythrophagocytosis by synovial cells. In their experiment using adult rabbits given a single injection of autologous blood, the following light microscopic changes correlated 10/ well with those of Key: there were red cells and hemosiderin within synoviocytes by four hours post 41 injection; an acute inflammatory reaction (neutrophilic) peaked by day four and largely subsided by day seven; synovial membrane hyperplasia with villous formation peaked at day four, but was still evident at day seven; and the subintimal tissue was hypercellular (fibroblasts and macrophages) and contained numerous blood vessels giving it a granulation tissue appearance. Transmission EM verified erythrophagocytosis by synoviocytes. The phagocytized erythrocyte is initially compartmentalized by double 917 membrane partitions. It is speculated that this provides channels which permit hydrolytic enzymes to reach 7 2 the deeper parts of the phagocytized erythrocyte. The phagocytized erythrocyte may be further broken down by a process called 'tunnelization', where the erythrocyte is riddled with invaginations of the erythrophago1 ysosome, 7 2 giving it a moth-eaten appearance. Other types of lysosomal or residual bodies seen with hemarthrosis include: mye1 inosomes, which contain whorled osmiophilic membranes representing residual erythrocyte membranes; siderosomes, which contain electron dense hemosiderin
(iron-containing) particles; and myelinosiderosomes, which 7 2 21 7 are combinations of the above. * These structures are seen in acute hemarthrosis, but not as frequently as they are in chronic hemarthrosis.
Other changes in synoviocytes following hemarthrosis include a marked increase in the amount of endoplasmic 42 reticulum as well as dilation of cisternae of the 79 917 endoplasmic reticulum. Ghadially 1 interprets these as
type B and AB synoviocytes, and correlates it with increased hyaluronate production by these cells in response to the joint insult. In contrast to chronic hemarthrosis, there is no increase in intracytoplasmic filaments or lipid droplets in synoviocytes, or changes in chondrocytes associated with acute hemarthrosis. All of the changes described with acute hemarthrosis in experimental animals have corresponded well to the natural condition of 9 1 o traumatic hemarthrosis in man.
Chronic Hemarthrosis
Chronic hemarthrosis has been extensively studied primarily because of the role it plays in hemophilic arthropathy. Hemophilia in humans is a sex-linked recessive disorder, expressed in males and carried by females. However in hemophilic dogs, males are affected 259 more frequently but females are also affected. In humans there are two types of hemophiliacs that frequently develop hemarthrosis. Hemophilia A (Classic hemophilia) is caused by a deficiency of factor VIII, and hemophilia B
(Christmas disease) is caused by a deficiency of factor IX.
Bleeding tendency, and therefore the tendency for spontaneous hemarthrosis, is directly related to the degree of deficiency of the clotting factor.There is a high incidence rate for developing hemophilic arthropathy in 43 individuals with severe factor deficiencies. In these individuals, hemarthrosis begins in early childhood and progresses to debilitative hemophilic arthropathy by their
late teen years and frequently requires surgical treatment.
The lesions of human hemophilic arthropathy have been well studied, primarily because of the availability of tissues following prosthetic sur geries. * ^ ^ ^ > 193, 204,253,274 Initial hemarthrotic episodes are quickly removed from the joint, but removal is
less complete and slower with each subsequent episode.
Gradually the synovial membrane becomes discolored golden- brown and thickened. The discoloration is due to hemosiderin within synoviocytes and subintimal macrophages.
The thickening is due to synovial membrane hyperplasia and increased vascularity of the subintima. The subintima also contains moderate numbers of lymphoctyes, plasma cells and fibroblasts. Pannus formation begins at the margins of the articular cartilage. Eventually the articular cartilage also becomes discolored and softened. Erosions and ulcerations bare the subchondral bone. Subchondral bone cysts are common and frequently multiple. Occasionally massive periosteal or intraosseous hemorrhage creates large hematomas called 'hemophilic pseudotumors'. Due to their development beneath the periosteum, they frequently become ossified during organization and, grossly and radiographically appear as boney growths. Hemophilic arthropathy in animals rarely is seen, for these individuals do not survive long enough for its development without adequate medical management.
Swanton^^ studied hemophilic arthropathy in dogs by maintaining a colony of hemophilic dogs while providing factor replacement therapy. The study included pathologic descriptions of 34 dogs who spontaneously died from hemophilic complications. All dogs developed recurrent lameness by the first year of life. Thirteen of 15 dogs greater than three years old had moderate to severe joint disease. Swanton concluded that canine hemophilic arthropathy is essentially identical, clinically and pathologically, to that of humans.
The ultrastructural changes in chronic hemarthrosis 7 S differ markedly from that of acute hemarthrosis. There is marked synoviocyte hypertrophy and hyperplasia, due to
■7 C O C O increases in type AB and type B cells. ’ Normal type A cells are rare. Moderate to severe dilatation of the RER is a common feature as it is in acute hemarthrosis. But in contrast to acute hemarthrosis, synoviocytes in chronic hemarthrosis frequently have marked increases in
■7 C 1EO intracytoplasmic filaments. ' This is interpreted as a nonspecific degenerative change. One of the most striking features in the synovium is the presence of numerous R 1 siderosomes in virtually every synoviocyte. There are also multiple small electron-dense iron-containing 45 particles occasionally seen in the cytoplasm. The intima Q I may also be thickened by an abundance of matrix
Subintimal macrophages and fibroblasts are numerous.
Macrophages also contain numerous solitary and compound siderosomes.
There appears to be some species variability in the changes seen in chondrocytes from hemophilic cartilage.
Hough et a l ^ ^ examined hemophilic cartilage from five humans and two dogs. He found increased amounts of RER, decreased numbers of mitochondria, and numerous electron- dense iron-containing particles in most chondrocytes.
However, he only found siderosomes in one human specimen and none in the dogs. Electron probe x-ray analysis demonstrated Fe and P in the siderosomes. ncq Stein and Duthie looked at cartilage from 39 humans with hemophilic arthropathy and described: increased RER with and without cisternal dilation; swollen and fragmented mitochondria; increased intracytoplasmic filaments in the perinuclear zone; small Golgi complexes; and electron-dense iron-containing particles and siderosomes in the cytoplasm.
They concluded that all iron-containing chondrocytes were in varying stages of necrosis.
Experimental chronic hemarthrosis in rabbits has been p z: 01 A studied by Ghadially and Roy ’ and typical chondrocyte changes included: chondrocytes with siderosomes in every animal; abundant amounts of glycogen and intracytoplasmic 46
filaments; dilatation of RER; a paucity of Golgi complexes
and secretory vacuoles; a modest increase in lipid
droplets; and an overall increase in the number of necrotic
chondrocytes. They found siderosomes initially in the
tangential and transitional zones with deeper chondrocytes
involved only as the experiments progressed with multiple
injections. Electron probe x-ray analysis confirmed the
presence of Fe and P within the siderosomes.
Species variability in hemarthrosis is also apparent
in the general susceptibility of articular cartilage to
degenerative changes. This has been verified in animal
experiments designed to produce chronic changes similar to
hemophilic arthropathy. In the rabbit and dog, it takes
several injections of blood over a prolonged time period to
produce degenerative chanes in the articular
cartilage. 112,216 jn a stU(jy with rabbits, ultrastructural
changes only were present after six months of once-weekly
9 1 intra-articular injections of autologous blood. In a 119 study with dogs, only superficial fibrillation was
produced after 12-18 weeks of six-times weekly injections.
This is in marked contrast to an experiment done using
1 9 young adult Shetland ponies. In that experiment, the
stifle and carpal joints were filled with autogenous blood
and horses were sacrificed at 15 days, one, two and three months post injection. A typical hyperplastic and
inflammatory synovial membrane reaction was present in all 47 but the three month group. Most significantly, there was loss of metachromatic staining, fibrillation, erosions, and chondrocyte degeneration in the three month group of horses. There were no lesions in control joints receiving saline or serum. This would indicate an apparent increased susceptibility of horses to the harmful effects of intra- articular blood; in particular the production of cartilage lesions.
Mechanisms of Hemophilic Arthropathy
Since chronic hemarthrosis is invariably associated with synovial membrane hyperplasia, enzymatic mechanisms are obviously important in the pathogenesis of cartilage destruction. All enzymes discussed previously under inflammatory and noninflammatory arthritis could play potential roles in degradation of the cartilage matrix. It has been shown that increased levels of hydrolytic enzymes are present in the synovial fluid of hemophiliacs,^ and that the hemophilic synovial membrane can produce large 135 amounts of collagenase, neutral proteases and lysozyme. 9 1 Also the ultrastructural study by Roy suggests that there may be an intracytoplasmic escape of lysosomal enzymes during attempts by the chondrocyte to degrade siderosomes.
The unique factor to consider in hemophilic cartilage destruction is the role of iron as a potential cytotoxin.
The presence of Fe and P has been verified by electron 901 probe microanalysis in siderosomes of chondrocytes and
synoviocytes.®^ How iron precisely arrives at its
intracellular location within chondrocytes is
controversial. Hemoglobin is generally considered too
large a molecule to diffuse through cartilage matrix,
but may be able to in fibrillated cartilage. However, Choi 3 9 et al believes that smaller iron-containing compounds are more likely. Choi also showed that unbound ionized iron
(Fe++ and Fe+++) was cytotoxic to chondrocytes. The toxicity of iron compounds has been demonstrated previously in immature rabbits given intramuscular injections of iron 9 / dextran. In this experiment there was decreased metachromatic staining, fibrillation, and chondrocyte death evident in the articular cartilage. Further evidence of chondrocyte insult by iron compounds is suggested by
Kirkpatrick et a l ^ ^ who demonstrated decreased proteoglycan synthesis in chondrocyte cell cultures exposed to hemoglobin, ferritin, ferrous chloride and ferric chloride.
The above evidence for direct chondrocyte toxicity by iron containing compounds detracted from some older theories of cartilage destruction in hemarthrosis; in particular, the theory that iron compounds reacted with sulfated proteoglycans to form a salt in the matrix,It was reasoned that the salt created an abnormal and weak matrix which was susceptible to shear forces during weight 49 bearing. Another point against this theory is that ultrastructural studies have consistently failed to reveal significant amounts electron-dense iron-containing particles in the matrix.
The exact mechanism by which iron produces its cytotoxic effects on chondrocytes has not been determined.
Ferrous iron has a high oxidation potential and could possibly produce irreversible oxidation of respiratory enzymes in mitochondria or oxidation of lysosomal membranes and subsequent intracellular release of their hydrolytic enzymes. Both possibilities have some support as noted previously.
A potential role of iron in the pathogenesis of chondrocalcinosis has been suggested, for iron is capable of inhibiting pyrophosphatase enzymes which could enhance the precipitation of calcium pyrophosphate crystals,
Chondrocalcinosis has been reported in patients with hemophilia,119,255 50
Calcium Pyrophosphate Dihydrate-Deposition Disease
Crystal-Associated Arthropathies
The list of crystal-associated arthropathies has steadily increased over the last several years.53,194 Qout
(monosodium urate) crystals were the first to be recognized 907 as a distinct clinical entity, but now other common crystals include calcium pyrophosphate dihydrate
(pseudogout), calcium hydroxyapatite (apatite), calcium hydrogen phosphate dihydrate (brushite) and cholesterol,^*The apatite, brushite, and pseudogout crystals are closely related crystals and are collectively referred to as the calcium phosphate crystals. It was initially assumed by most investigators that crystals were directly responsible for pathologic changes found in the joint, but this viewpoint is now being questioned. In many crystal-associated arthropathies it is not known whether crystals produced the joint disease, or if joint disease produced the crystals, or if some uknown factor interplayed in either direction.^ This has led to the establishment of criteria, other than the mere intra-articular presence of the crystal, for the diagnosis or crystal-induced arthropathy. The criteria are: the arthropathy be a well defined clinical syndrome; crystals be consistently associated with the syndrome; the crystal type be definitively identified, and the injection of crystals can reproduce the arthropathy. 51
Once crystals have been located in articular tissues, a combination of analytical techniques is usually needed to
definitively identify the crystal type. Compensated
polarized light microscopy provides a rapid and accurate method for distinguishing urate crystals from the calcium
phosphate crystals. Urate crystals are strongly, positively birefringent and calcium phosphate crystals are weakly, negatively birefringent. Ultrastructural techniques have also been useful in determining morphological differences between crystals. Scanning EM is most useful in determining the crystal habit (shape and dimensions), but transmission EM can provide the cross- sectional shape. Urate crystals are long and acicular, whereas pseudogout crystals are plate or flake-like. To determine the finer differences between the calcium phosphate crystals, more sophisticated analytical techniques must be used. * An energy dispersive x-ray fluorescence spectrometer (EDX) attached to scanning electron microscopes can detect, map and relatively quantitate elements in the crystal. This method can be used to distinguish hydroxyapatite crystals (Ca:P = 1.67) from brushite and pseudogout crystals (Ca:P « 1.0).
Another electron microscope accessory can be used to differentiate the three types of calcium phosphates by x- ray diffraction patterns. These patterns are made when x- rays are passed through a crystal. Each crystal has a 52 characteristic diffraction pattern (fingerprint) enabling definitive identification of the crystal type.^**^*^^
Pyrophosphate Arthropathy
Pyrophosphate arthropathy is the term utilized by one Resn'ick and Niwayama to describe the pattern of joint damage that occurs in calcium pyrophosphate dihydrate deposition disease (CPPD-DD). Due to its occasional clinical similarity to gouty arthritis (intermittent acute arthritic attacks), the disease has also been referred to as pseudogout. The disease affects both sexes and is usually first seen in middle-aged and elderly patients.
CPPD-DD is by far the most common crystal-induced arthropathy of the elderly.The disease has been divided into three categories: hereditary; associated with other diseases; and idiopathic. The vast majority of idiopathic cases are found in elderly patients, and may actually be associated with osteoarthritis, but it is unknown whether the crystal produced degenerative joint disease or if the reverse is the case. The strongest evidence of association with other disorders exists for one primary hyperparathyroidism and hemochromatosis.
There are currently six clinical patterns described for CPPD-DD in humans which include: the pseudogout pattern, characterized by acute attacks with the knee being the most common site; pseudo-rheumatoid arthritis is characterized by continuous acute attacks; pseudo- 53 osteoarthritis, characterized by chronic progressive arthritis; pseudo-osteoarthritis with acute exacerbations; and asymptomatic deposition, which may be the largest 20'5 category. J
The gross cartilage changes of pyrophosphate arthropathy are essentially indistinguishable from severe 1 L 7 osteoarthritis, * Multiple erosions, ulcerations, eburnation, subchondral bone sclerosis, bone cysts, and marginal osteophyte formation are common findings in the articular cartilage. The synovial membrane may show varying degrees of thickening and reddening. Crystal deposits are seen on both the articular cartilage and synovium as a chalky white deposit of varying intensity.
Microscopically, fibrillation, cleft formation, chondrocyte cloning, and minimal metachromatic staining are common in areas of severe cartilage degradation; as well as decreased metachromatic staining in other less involved areas.Crystal deposits generally appear as delineated round areas in the transitional or radial zones of the articular cartilage. 19,201 uitrastructurally, no intracellular (chondrocyte) crystals are found and the crystals appear to originate in the terrirorial or pericellular matrix.4,19,201 Although some chondrocytes have nonspecific degenerative changes, chondrocytes in most studies are essentially normal. However, in the recent report by Ali et al / who was the first to describe 54
crystals 'within' lacunae, chondrocytes had increased
amounts of RER and glycogen indicative of increased or
deranged metabolic activity. Crystals seen by transmission
EM are moderately electron dense, frequently artifactually
fragmented and found in the matrix. The crystals
frequently form vacuoles and bubble while examining them
due to deterioration associated with vacuum pressures.
Occasionally the crystalline material will be lost during
thin-sectioniing and 'ghost' outlines (rectangular and rod
shaped) are present in the matrix as clear empty spaces.
Scanning EM studies reveal varying amounts of crystal
deposits both on the articular cartilage and synovial
surfaces. Crystals in the form of plates, rods, rectangles 2Q 20ft and squares may be seen.
The microscopic changes in the synovial membrane are
also variable.The inflammatory response is mild to
moderate and consists of lymphocytes, plasma cells and
histiocytes. Infrequently the response can be marked and
more neutrophilic. Occasionally histiocytes surround
crystal tophi that are embedded in the intimal matrix, in a
foreign-body type reaction. Multinucleated giant cells are
rarely seen. Crystals are rarely present in histiocytes or
multinucleated giant cells. Scanning EM reveals crystals
on the synovial surface which may be partially covered by
proteinaceous material. Significant hemosiderosis was noted in two of twelve human cases in the study by Markel CPPD-DD is primarily a disease of man and has rarely
been reported in animals. One case of crystal deposition
go in the synovial membrane of a dog has been reported.
Familial calcium hydroxyapatite deposition disease has been
reported in Great Danes, but this is not the same crystal
as that of CPPD-DD.Other animal cases have been
limited to nonhuman primates. One reported case involved a
20-year-old female Barbary ape (Macaca sylvanus). The ape
had CPPD crystalis in multiple joints, intervertebral discs,
ligaments and menisci,Roberts et al^®® has reported
six cases of naturally occurring CPPD-DD in rhesus monkeys
(Macaca mulatta) and have found the disease in monkeys very
similar to that of humans. They have suggested that the rhesus monkey would be an appropriate animal model of the human disease for pathogenesis studies.Further support for the rhesus monkey as an animal model for CPPD-DD comes
from Pritzker (Personal communication with E.D. Roberts) who has recently identified naturally occurring CPPD-DD in rhesus monkeys from another primate center, the Caribbean
Primate Research Center in Puerto Rico.
Pathogenesis of Pyrophosphate Arthropathy
Although the inflammatory reaction in CPPD-DD is usually only mild to moderate and occurs secondary to the shedding of crystals into the joint space, it does account for a significant proportion of the cartilage destruction 56
that accompanies this disease. The inflammatory
mechanisms^® are similar to those described previously
under inflammatory joint disease and osteoarthritis.
Phagocytosis of crystals by neutrophils is followed by the
release of chemotactic factors by the neutrophils which
rapidly amplify the inflammatory reaction.It has been
shown experimentally that neutrophil depletion or
neutrophil protease inhibition can significantly inhibit
9 51 the inflammatory response. A The same study also showed
that complement was not necessary for the inflammatory
response to occur, as is the case in gout. Shed CPPD
crystals are taken up by synovial and inflammatory cells
and cleared at a rate inversely proportional to crystal
size. * Neutrophils that had phagocytized calcium
phosphate crystals (apatite) released greater levels of B-
Glucuronidase than those that phagocytized urate crystals
or non-crystalline particulate matter.Cultured synovial
cells have also been shown to release collagenase, neutral
protease and prostaglandins following phagocytosis of CPPD 07 crystals. Using lumino-dependent chemiluminescence to
assess the release of harmful oxidizing agents following
crystal phagocytosis has also been studied.Urate
crystals were shown to release five times more oxidizing agents than the calcium phosphate crystals (CPPD, brushite, apatite). Prostaglandins (PgE2) have also been shown capable of mediating the inflammatory reaction of CPPD-DD, 57 for cultured canine synovial fibroblasts released significant levels of PgE2 when exposed to CPPD crystals in concentrations comparable to that found in human synovial 1 c c oo fluid. Using the same culture system, Cheung et al also showed CPPD crystals are mitogenlc which may explain in part the synovial membrane hyperplasia seen in some 1 97 cases of CPPD-DD. Kozin and McCarty have shown specific synovial fluid proteins adsorb to intra-articular CPPD crystals, and among these are IgG. They speculate that this is necessary before neutrophils can phagocytize the crystals; there are Fc receptors on the surface of neutrophils. They also suggest that the 'suicide bag release phenomenon' does not occur until the protein coat is digested within the phagolysome resulting in exposure of the sharp edges capable of rupturing biological membranes.
The question of how and why crystals form in the crystal-associated arthropathies is still to be elucidated.
It is generally accepted that an increase in various solute concentrations and variable factors which stimulate and inhibit crystalization of these solutes, are responsible for the numerous different types of crystals found in arthropathies. The same factors could explain the various disease associations found in crystal-induced arthropathies. It is probable, that the many distinct syndromes, such as CPPD-DD, may actually represent the end- stage manifestation of several different pathogenetic 58 mechanisms which share a final common pathway. There are a variety of metabolic disturbances that lead to CPPD-DD^
Many of these are related to defects in or interference with pyrophosphate metabolism. Inorganic pyrophosphate
(PPi) generation' occurs during the biosynthesis of many bilolgical compounds including proteins, lipids, phospholipids, nucleotides, nucleic acids, steroids, urea, polysaccharides and g l y c o g e n . 222,224 Breakdown of PPi is by the hydrolytic action of inorganic pyrophosphatase enzymes such as glucose-6-phosphatase and alkaline phosphatase.32,222,224 These intracellular enzymes are specific for PPi, have an optimum pH of 7-8, require Mg++ as a cofactor and are strongly inhibited by divalent metal cations such as Ca++, Fe++, and Cu++,65,66,158,159,199,268
Two molecules of soluble orthophosphate (Pi) are produced per molecule of PPi cleaved by pyrophosphatase enzymes.
Recent studies have shown there is another enzyme capable of playing an important role in CPPD-DD.
Nucleoside triphosphate (NTP) pyrophosphohydrolase has been identified as a membrane bound ectoenzyme of chondrocytes and is capable of breaking down trinucleotides (such as
ATP) into nucleotide monophosphates plus ppi,32,115,176,225
It has been suggested that in cartilage, injured or degenerating chondrocytes may release nucleotides which can be acted upon by NTP pyrophosphohydrolase and lead to an increase PPi concentration in the pericellular matrix.A 59
similar mechanism can result from the action of other
membrane bound enzymes such as adenylate cyclase, which
splits ATP into 3’,5f-cyclic AMP plus PPi.^ Adenylate
cyclase can also be activated by certain hormones such as
parathyroid hormone.
Failure to breakdown PPi or excessive production of
PPi will eventually lead to high PPi levels accumulating in
tissues. High PPi levels and as yet unidentified factors
may then allow or influence the precipitation of insoluble
CPPD crystals. High levels of PPi have been identified in
synovial fluid from patients with CPPD-DD and patients with
osteoarthritis.223,242,243 gut apparent that high
PPi turnover and elevated synovial fluid PPi concentrations
are not specific for CPPD-DD alone and that other 28 influencing factors are necessary for crystal deposition.A
Recent studies using model hydrogels and aqueous
solutions have been done to determine ionic concentrations
and related factors that promote or inhibit crystalization of CPPD crystals.34-36,138,195,196 These studies have
shown that: optimum concentrations are 5-25 mM Ca and 1-4 mM PPi for triclinic CPPD formation; [Ca]/[Mg] and
[Pi]/[PPi] ratios are critical; cartilage proteoglycan is a potent inhibitor of CPPD formation; and that the crystals evolve from kinetic precursor intermediates.
As mentioned previously, CPPD-DD has many concurrent
disease associations. With the current state of knowledge 60 on pyrophosphate metabolism and factors affecting CPPD crystal formation, it is possible to speculate on the pathogenesis of crystal formation in some of these o n diseases:1-** in hemochromatosis and hemarthrosis, excess iron in cartilage may act as a nucleating agent or produce pyrophosphatase inhibition; in hyperparathyroidism, high calcium levels in the cartilage may act as a pyrophosphatase inhibitor, and parathyroid hormone may activate adenylate cyclase to increase PPi production; in hypophosphatasia, PPi is not catabolized due to a deficiency of alkaline phosphatase and PPi accumulates; in
Wilson's disease, excess copper in cartilage may act as a nucleating agent or inhibit pyrophosphatase; and in ageing, there may be disturbed PPi metabolism with increased age. MATERIALS AND METHODS
Experimental Animals and Procedures
Sixteen adult male rhesus monkeys (Macaca mulatta) were utilized in the study. The animals were mature and were acquired as juveniles during the late 1960's and early
1970's , with an approximate age between 11 and 18 years- old. The monkeys were healthy, well-fleshed and housed in individual cages within indoor facilities of the Delta
Regional Primate Research Center, Covington, LA.
Twelve of the monkeys were owned by the Department of
Pathology, LSU-Medical Center, New Orleans, LA and were on a simultaneous non-musculoskeltal system project to study the effects of atherogenic diets. Four of the monkeys were owned by the Delta Regional Primate Research Center and had been on various non-muscu1oskeltal system projects.
Therefore, a basic assumption of this hemarthrosis study was that the atherogenic diets that 12 of the monkeys were receiving would have negligible effects on this joint study. The validity of this assumption was based on: 1) degenerative joint disease has been an exhaustively studied subject and there has been no evidence to indicate dietary factors are significant in its pathogenesis, and 2) experimentation involved only one paired-joint therefore each animal had a contralateral control joint for comparison purposes.
The monkeys were randomly assigned to four groups of
61 62 four and were injected with autologous blood at various times to correspond with scheduled sacrifice dates. The four groups represented post-injection times prior to necropsy: Group 1 monkeys (Seven-day group) were sacrificed seven days post-injection; Group 2 monkeys (Two- month group) were sacrificed 50-60 days post-injection;
Group 3 monkeys (Three-month group) were sacrificed 82-85 days post-injection; and Group 4 monkeys (Six-month group) were sacrificed 183 days post-injection. The time groups were designed to allow any degenerative changes, stimulated by the joint insult, ample time to progress (or resolve) in efforts to observe early chondrocalcinosis.
Differences between principal and control joints were analyzed by T-pair statistical methods. Probability values were determined for both overall treatment differences
(n = 16) and for differences within each group (n=4).
Differences among groups were analyzed by one-way analysis of variance. Statistical calculations were done with an
IBM Personal Computer (IBM Corp., Boca Raton, FL) using an
ABSTAT statistical program (Anderson-Bel1 Co., Canon City,
CO) .
In jection Procedure
The monkeys were anesthetized with ketamine-HCl prior to removal from their cages. The femorotibial joints were examined and palpated to detect any pre-existing clinical abnormalities. The left femorotibial joint was shaved and 63 scrubbed with a povidone iodine surgical scrub, and rinsed with ethyl alchol. Blood was drawn from a site similarly prepared from the femoral venous plexus located high in the medial thigh. Three cubic centimeters (cc) of blood were drawn with a 20-gauge, 3/4 inch hypodermic needle into a 6- cc plastic syringe with no anticoagulant. The autologous whole blood was immediately injected into the prepared knee using aseptic techniques to avoid intra-articular contamination. The blood was injected through the articular capsule by palpating the patella, and passing the needle just ventral to the patella through the medial parapatellar fossa, until the femoral condyle was engaged.
The needle was then withdrawn slightly and the blood was injected. Confirmation of joint filling was by observing lateral and medial bulging of the articular capsule. The monkeys were then returned to their cages.
Collection Procedures
The monkeys were anesthetized with ketamine~HCl and an abdominal incision was made. The caudal vena cava was catheterized and the monkeys were nearly exanguinated and then euthanized by an intravenous injection of sodium pentobarbital. The femorotibial joints were immediately amputated by exposing the distal femur and proximal tibia and severing them with an electric autopsy saw. The severed joints were placed in a vice while tissue samples were collected. Prior to opening the articular capsule, 64
3cc of sterile saline were injected into the joint and withdrawn as a joint rinse. The joints were cultured immediately after opening to detect any contamination that may have occurred during the injection procedure.
For biochemical and metabolic studies, articular cartilage (10-30 mg dry weight) was removed from the proximal patellar groove using aseptic techniques and immediately placed in culture media and held at body temperature.
Selected tissues for electron microscopy were removed and placed in 2% glutaraldehyde in ,1M sodium cacodylate.
The remaining joint was placed in a fixative that contained
10% formalin and 1% glutaraldehyde.
Light Microscopy
Tissues trimmed for light microscopic evaluation were: synovial membranes from the anterior perimeniscal region of the lateral meniscus, the patellar fat pad, and the proximal trochlear groove; a cross section of meniscus from the lateral side of the lateral meniscus; femoral articular cartilage and bone from the weight bearing and nonweight- bearing surfaces of the lateral condyle; tibial articular cartilage from the lateral condyle; extensor skeletal muscle (quadriceps femoris); and flexor skeletal muscle
(gastrocnemius).
Following fixation for at least 72 hours, tissues were processed routinely for paraffin embedding, sectioned at 4- O 7 6 urn and stained with hematoxylin and eosin. Bone
sections were demineralized prior to sectioning with 10% formic acid citrate decalcifying solution under vacuum until sections were decalcified enough to be trimmed easily with a razor. Sections of articular cartilage were also stained with Safranin 0 and counterstained with Fast Green and Hematoxylin.125,215
Weight-bearing articular surfaces from the lateral femoral condyle were evaluated according to the Mankin osteoarthritis grading system (Fig. 1).*^
Electron Microscopy
Scanning Electron Microscopy
Tissues prepared for scanning EM evaluation were: synovial membrane from the anterior perimeniscal region of the medial meniscus and the patellar fat pad; articular cartilage from the weight-bearing medial femoral condyle and tibial condyle; and the medial meniscus.. Following fixation at 4° C in 2% glutaraldehyde in .1M Na-cacodylate for 1-2 weeks, tissues were stored at 4° C in ,5% glutaraldehyde in ,1M Na-cacodylate with 5% glucose until further processing. Tissues were then rinsed with .1M Na- cacodylate buffer with 5% sucrose (3 X 10 minutes); post fixed in 1% osmium tetroxide in .1M Na-cacodylate buffer for 60 minutes; rinsed (3 X 10 minutes) in distilled water; dehydrated in a graded ethyl alchol series; and dehydrated in a CO2 critical point dryer. Samples were then mounted 66
Mankin Osteoarthritis Grade
I. Structure a. normal b. surface irregularities c. pannus and surface irregularities d. clefts to transitional zone e. clefts to radial zone f. clefts to calcified zone g. complete disorganization
II. Cells a. normal b. diffuse hypercellularity c. cloning d. hypocellularity
III. Safranin-0 Staining a. normal b. slight reduction c. moderate reduction
d. severe reduction W M ^ O M to LO O U W ■(> H L/l O O' e. no dye noted
IV. Tidemark Integrity a. intact 0 b. crossed by blood vessels 1
Total Score
Fig. 1. Mankin osteoarthritis grading system. 67 with colloidal carbon and sputter coated with gold- palladium (80:20) for eight minutes. The tissues were examined with a Cambridge Stereoscan S150 II scanning electron microscope (Cambridge Instruments, Cambridge,
England) .
Transmission Electron Microscopy
Tissues examined by transmission electron microscopy include the synovial membrane from the anterior, perimeniscal region of the lateral meniscus and weight bearing articular cartilage from the medial femoral condyle. Tissues not processed immediately following the initial glutaraldehyde fixation were stored as noted above for SEM tissues. Tissues were then post-fixed in 2 % osmium tetroxide; rinsed in 0.1M Na-cacodylate (3X5 minutes); dehydrated in a graded ethyl alchol series; placed in propylene oxide (3 X 10 minutes); and embedded in Epon-
Araldite epoxy resin. Following polymerization and thin- sectioning, samples were stained 30 minutes with uranyl acetate and 5 minutes with lead citrate. Tissues were examined with an EM 10 A/B transmission electron microscope
(Carl Zeiss, Inc., Oberkochen, West Germany).
Ultrastructural Enzyme Localization
Tissue samples of synovial membrane from the anterior perimeniscal region of the lateral meniscus and weight bearing articular cartilage from the medial femoral condyle were processed for localization of acid and alkaline 68 phosphatase, immediately after initial fixation in glutaraldehyde for 3-4 hours. Two methods were employed, each utilized heavy metals (either lead or cerium) to trap phosphate ions at sites of release.^’^ ’^ ^ In both methods, tissues were incubated 60 minutes in media that contained B-glycerophosphate as the phosphatase substrate and 0.2% lead nitrate or 2 mM cerium chloride respectively.
Localizations were done prior to post-fixation in osmium tetroxide and then processed as described. Negative controls used were no substrate controls and inhibitor controls (NaFl and Tetramisole). Liver from the same animal was used as a positive control tissue. Both stained and unstained sections were examined for electron-dense heavy metal particles with the transmission electron microscope.
Biochemical and Metabolic Analyses of Articular Cartilage
Isotope Labeling and Initial Processing
The harvested cartilage was initially incubated at 37°
C for 24 hours in equilibration media (Fig. 2) composed of
9 ml of Ham^l2 culture media, 1 ml of calf serum, 50 ug/ml ascorbate and 30 ug/ml ketog1utarate. The cartilage was aseptically transferred to 10 ml of the same type of media ft with isotopes added (25 uCi/ml H-proline and 25 uCi/ml
Na2_^^So^), (ICN Biomedicals, Inc., Irvine, CA), After exactly 18 hours incubation with isotope, cartilage biosynthesis was stopped by the addition of protease 69
Double Labeling Procedure
Protease Inhibitors
Freeze
Necropsy \ $ y v i z W Culture Culture Media Stop Media and Isotopes Reaction 24hrs. 18hrs. Z 7 ° C 37°C
Fig. 2. Isotope labeling procedure for the metabolic study of articular cartilage.
Biochemical Analyses
Total Protein Assay
2N HC1 .Hydroxyproline Assay
Hexosamine Assay■ W w Thaw Save Lyophllize Hydrolyze Forced Air Media 4Shrs. 12hrs./l 10®C Evaporator for Count
Fig. 3. Initial processing of articular cartilage for biochemical analyses. 70 inhibitors composed of ,2M EDTA, .1M N-ethylamleimide, .1M benzamine HC1 and ,5mg/ml phenylmethylsulfluoride. The samples were then frozen at -60° C and held until further processing.
Samples (Fig. 3) were then thawed and transferred from the incubation media to plastic test tubes and dehydrated for 48 hours in a CO2 tissue lyophilizer., The incubation media was saved and an isotope count made to detect secreted products. Between 10-12 mg of cartilage (dry weight) from each sample were weighed on a Cahn 21
Automatic Electrobalance (Cahn Instruments Inc., Cerritos,
CA) The lyophilized cartilage was then hydrolyzed for 12 hours at 110° C in 2N HC1, and dryed in a Meyer N-Evap
Analytical Evaporator (Organomation Assoc. Inc.,
Northborough, MA). The samples were then divided for three separate analyses; total protein assay, hydroxyproline assay and hexosamine assay.
Total Protein Assay
The total protein assay was performed using the 1 methods described by Lowry et al. The basis of the reaction can be explained in two steps (Fig. 4): protein binds to copper in an alkaline solution and the copper- protein complex reduces the Folin reagent. After 30 minutes, absorbance was read at 750 nm on a Beckman Model
35 Spectrophotometer (Beckman Inst. Inc., Palo Alto, CA),
Results in ug/mg of articular cartilage were determined 71
Total Pfotatn Assay
1m)
10 mln, + 100ul Folln
Somplo
30 mTm a* 7S0nm -s?
Fig, 4. Procedure for the total protein assay of articular cartilage.
Hesoaamlno Aaaav
SH MCI 2 m Ma,C0,
H,0 V — / v_y Sampla Neutralise ' Bolling HjO 20 mln. 1ml Ethanol Ehrllch’e Reagent
5 mln. „v lee O' S30nm v_y
Fig, 5. Procedure for the hexosamine assay of articular cartilage. 72
from standard curves made from bovine serum albumin treated
identically for each run of the assay.
Hexosamine Assay
Hexosamine is an integral component of
glycosaminoglycans. Therefore its assay is a direct
estimation of proteoglycan content of the articular
cartilage. The procedure used (Fig, 5) was that described
by Gatt and Berman.^ The hydrolyzed samples were
neutralized with sodium carbonate and then boiled with
acetyl acetone and cooled in ice. Chromatophore formation
occurs when hexosamine reacts with the p-
dimethylaminobenzaldehyde present in Ehrlich's reagent.
Absorbance was read at 530 nm. Results were expressed in ug/mg articular cartilage as determined from standard curves made from purified glucosamine controls treated identically for each run of the assay.
Hydroxyproline Assay
Hydroxyproline is an integral component of collagen and therefore its assay is a direct estimation of collegenous protein in articular cartilage. The procedure used (Fig, 6) was that described by Switzer and Summer.
Following the initial hydrolysis as described above, the samples were hydrolyzed a second time in stronger (6N) hydrochloric acid for 3 hours at 130° C. Samples were then oxidized with chloramine-T solution for 25 minutes, and the oxidation was stopped by adding thiosulfate. The solution 73
Hvdroxyprollno Assay
6N HCI Chloramlno-T Thlosulfolo KCI
Neutralize V -" Count
W w Sample 2nd Hydrolysis Oxidation Stop Oxidation 3hrs./130°C
Bolt Aqueous Discard Layer tor Shake Toluene 30 mln. Layer (Pyrrolo)
Toluene
Centrifuge
Shake
Ehrttch’s Reagent Silicic Acid Count Column tju 30 mln. 560nm *
Fig. 6. Procedure for the hydroxyproline assay of articular cartilage and sampling points for metabolic studies. 74
was then saturated with potassium chloride and toluene was
added to extract the proline oxidation product and other
interfering compounds. Hydroxyproline was then converted
to pyrrole by heating in a boiling water bath for 30 minutes. Toluene was added again, this time to extract
pyrrole. The pyrrole was then reacted with Ehrlich's
reagent and quantitated by reading absorbance at 560 nm on
a spectrophotometer. Results were expressed in ug/mg of
articular cartilage and were determined by comparison to
standard curves made from purified hydroxyproline treated
identically for each run of the assay.
Metabolic Assay
The double-labeling procedure was described above
(Fig. 2). Three parameters were determined from samples removed and counted at different times during the hydroxyproline procedure (Fig, 6): 1) cartilage total protein, 2) collagenous protein and, 3) proteoglycan
synthesis in 18 hours. All isotope counts were done on a
Beckman-LS250 liquid scintillation counter (Beckman Inst.
Inc., Palo Alto, CA). Cartilage total protein production was determined by doing a count for H on a sample of hydrolyzate after the second hydrolysis. Cartilage collagenous protein production was determined by doing a count for H on a sample from the hydroxyproline assay after passing the toluene extract through a silicic acid column. The silicic acid column removes non-pyrrole 75
labeled compounds (non-collagenous proteins) from the
*3 extract so that H counts then represent pure collagenous
protein synthesis. Counts for SO^ performed after the
second hydrolysis represent cartilage proteoglycan
production.
Counts were also done on the cartilage culture media following three days of dialysis in 3,500 molecular weight cut off membrane tubing. This was to determine the cartilage total protein (^H) and proteoglycan (^SO^) production that was secreted into the media.
Morphometric Analyses
Weight-bearing femoral articular surfaces from the
lateral femoral condyle were analyzed from both left and right knee joints. Morphometry was done with an IBM
Personal Computer (IBM Corp., Boca Raton, FL) with an IBM
Bone Morphometry Program (R & M Biometrics, Nashville, TN), using 4-6 um demineralized paraffin embedded sections stained with hematoxylin and eosin. Parameters determined for articular cartilage were: matrix-rcell area ratios; Q cellularity (cells per mm ); per cent of cartilage occupied by lacunae; and average lacunae area (um ). Parameters determined from subchondral bone were: per cent of area occupied by trabecular bone, and osteocyte lacunae area expressed as per cent of trabecular area.
Synovial membrane from the anterior perimeniscal region of the lateral meniscus was also evaluated 76 raorphometrically. Parameters determined were: cellularity
(cells per linear mm) and average intimal thickness (mm). RESULTS
Morphologic Observations
The injected blood was not visible in the principal joints except in one animal from the 7-day group. In that knee, there were three small clots (3mm across) free in the joint space. The synovial membranes from 9 of 16 principal joints contained multiple patches of yellow-brown discoloration (3x5 mm), especially involving the anterior perimeniscal region and the patellar fat pad.
Discoloration involved all groups, but was most prominent in groups 1 and 2. The control joint synovial membranes were grossly normal in appearance.
There were no differences between principal and control joints in the appearance of the articular surfaces and menisci. Severe osteoarthritis was present in one control knee. In that joint there were multiple erosions and ulcers of both femoral and tibial articular surfaces.
Many osteophytes were present along the lateral trochlear ridge and the lip of the lateral tibial plateau. A few subchondral bone cysts were also present. The lateral meniscus was obliterated.
Bacterial cultures of all joints were negative after
72 hours incubation on blood agar plates.
Microscopic Evaluation
Articular Cartilage-Light Microscopy
77 78
Utilizing the Mankin osteoarthritis grading system,
each weight-bearing femoral articular cartilage specimen was given a numerical score* The overall average score of
the principal joints was not significantly different from
that of the control joints (Table 1), The structure of the
cartilage generally scored normal or there were minor
surface irregularities. Severe structural changes were
only noted in the severely osteoarthritic control joint
from group 4 previously described under gross observations.
In that joint there was fibrillation, numerous clefts and focal areas of complete disorganization. There was considerable cartilage cellularity variation between animals. Several animals had cloning or hypocellularity which was general or marginal in location. Tidemark integrity was intact in all specimens,
Safranin 0 staining affinity was decreased in the principal joints, especially evident in group 1 and 2 principal joints. Safranin 0 staining affinity was generally poor in the tangential zone and at the edge of the articular plate, where cartilage blended into fibrocartilage and the periosteum. Staining affinity generally decreased starting from the tangential zones and extended into the deeper cartilage zones. Staining affinity relative to the cartilage matrix zones generally decreased in the following order: territorial matrix, interterritorial matrix and pericellular matrix. 79
Nonweight-bearing femoral articular cartilage and tibial articular cartilage were normal. Approximately one- third of each medial tibial plateau was composed of fibrocartilage. This area stained positively with Safranin
0 only in the deeper layers. The remainder of the tibial articular surface was composed of hyaline cartilage which gradually thinned toward the periphery of the tibial plateaus. Safranin 0 staining affinity decreased where the hyaline cartilage was thinner.
Articular Cartilage-Transmission Electron Microscopy
Mild morphologic changes were present in group 1 chondrocytes from the tangential and transitional zones.
These include an increase in size and number of cytoplasmic vacuoles, especially prominent at the periphery of the cell
(Fig. 7). Some vacuoles appeared to be formed by long extensions of cytoplasmic processes and incorporation of portions of pericellular matrix (Fig. 8). These vacuoles contained electron dense material similar to that of the pericellular matrix. There were also increased numbers of myelin figures and residual bodies present in tangential and transitional zone chondrocytes of group 1 (Fig. 9).
Myelin figures were composed of concentric layers of membranes surrounded by electron dense flocculent material and granules. Lipid droplets surrounded some residual bodies and in some instances appeared to be discharging lipid contents into them (Fig. 10). Rarely, single Fig. 7. Group 1 chondrocytes from tangential and transitional zones of principal joints. A) Vacuolation was especially prominent at the cell periphery. Bar = 1 um. B) Cytoplasmic vacuolation and lipid accumulation. Note long cytoplasmic processes. Bar = 1 um. Fig. 8. Transitional zone chondrocyte with long cytoplasmic processes and peripheral cytoplasmic vacuolation. Bar= 1 um. 82
Fig. 9. Variety of myelin figures found in tangential and transitonal zone chondrocytes from group 1 principal joints. A) Bar = .25 um. B) Bar = .1 um. C) Bar = .25 um. 83
Fig. 10. Group 1 principal chondrocytes. A) Lipid droplet apparently discharging contents into cytoplasmic vacuole that contains myelin figures and cell debris. Bar = .2 um. B) A single membrane bound body containing electron dense granular material in a group 1 principal chondrocyte. Bar = .5 um. 84
membrane bound bodies containing electron-dense granules were seen and interpreted as siderosomes (Fig. 10),
Chondrocytes in radial zones were normal.
Chondrocytes from groups 2, 3 and 4 were essentially
similar when principal and control joints were compared.
Radial zone chondrocytes had well developed Golgi complexes and rough endoplasmic reticulum. Lipid droplets were common and in many cells occupied a large portion of the cytoplasm (Fig. 11). Large accumulations of intracytoplasmic filaments were seen in some cells, usually in the perinuclear position (Fig. 11). The matrix was essentially normal, composed of collagen fibers and proteoglycan particles.
Crystals were rarely seen in the matrix of both principal and control articular cartilages. Some individual crystals were square or rectangular, morphologically compatible with CPPD crystals (Fig. 12).
However, the crystals were too small (less than ,5 um) to produce a diffraction pattern,for definitive identification.
Ultrastructural localization of acid and alkaline phosphatase were unsuccessful in both articular cartilage and synovial membrane.
Articular Cartilage-Scanning Electron Microscopy
There were no morphologic differences in articular surfaces between principal and control joints. The typical 85
Fig. 11. Normal variations in radial zone chondrocytes. A) Large cytoplasmic lipid droplets. Bar = 1 um. B) A swirl of intracytoplasmic filaments. Bar = .5 um. 86
Fig. 12. An example of small matrix crystals rarely seen in the cartilage of both principal and control joints. Bar = .2 u m . 87
femoral articular surface was relatively smooth, with evenly distributed "humps and pits". Variably sized round droplets of synovial fluid were adhered to the surface in many areas. Surface irregularities were occasionally seen, most were shallow erosions. A typical erosion was an area where the armor plate layer was absent, exposing a meshwork of collagen fibers (Fig. 13). The articular surface adjacent to an erosion was often folded and the armor plate was often cracked.
The tibial articular surface was similar to the femur, except for the roughened medial area of fibrocartilage
(Fig. 13). In that area the surface was consistently thrown up in large, irregular folds covered by a meshwork of large collagen fibers. In some areas, collagen fibers were markedly fragmented and frayed (Fig. 13).
Synovial Membrane-Light Microscopy
Changes in the synovial membranes from the four collection sites were relatively uniform within each joint
(anterior perimeniscus, posterior perimeniscus, patellar fat pad and proximal trochlear groove). The most significant changes were in the group 1 principal joints where all displayed synovial membrane hyperplasia. A description of a typical perimeniscal synovial membrane from a principal group 1 joint follows. The intima was three to six cell layers thick whereas a comparable control membrane was one to three cell layers thick (Fig. 14). Fig. 13, Normal variations found in the articular surfaces of the femur (A & B) and tibia (C & D). A) Erosion with exposure of collagen fibers and wrinkling of the articular surface. Bar = 200 um. B) Cracking and loss of the armor plate layer with exposure of collagen fibers in an erosion. Bar = 20 um. C) Abrupt change from smooth to rough surface in the medial tibial plateau. Bar = 150 um. D) Exposed and frayed collagen fibers from articular surface of medial tibial plateau. Bar = 20 um. 89
Fig. 14, Group 1 perimeniscal synovial membrane from principal (A) and control joints (B). A) Hyperplastic synovial intima with scattered subintimal inflammatory cells and perivascular cuffing (arrow). B) Normal synovial membrane from counterpart joint; note thin intimal layer and absence of subintimal inflammatory cells. 90
Cytoplasmic borders in principal synoviocytes were more distinct than the controls, giving them a generally larger appearance. Synovial villi were not increased in number but were more prominent and somewhat larger due to the thick intimal layer covering them. In some instances, adjacent villi were adhered to one another by fibrinohemorrhagic material (Fig. 15). There was a mild to moderate increase in mononuclear cells and occasional neutrophils in the subintima. Erythrophagocytosis was evidenced by macrophages in the subintima that contained erythrocytes in their cytoplasm (Fig.15). Macrophages laden with brown pigment were occasionally seen. The subintimal vasculature was more prominent in the principal membranes, some vessels were rimmed by lymphocytes and macrophages.
The synovial membranes in groups 2, 3 and 4 had essentially returned to normal, when comparing principal and control joints. Phagocytic cells laden with brown pigment were evident in the subintima of several of the principal joints, representing all of the later groups.
The brown pigment was interpreted to be hemosiderin, formed as a by-product of red cell degradation. The amount of pigment found generally decreased with time. Two principal joints in group 3 and one joint in group 4 had no pigment evident. Similar pigments were sometimes found in control joints as well but always to a lesser degree than the 91
Fig. 15. Synovial membrane from group 1 principal joints. A) A large clot formed by red cells (R) and fibrin (F) adhered to the intima (arrows). B) Hypercellular area of subintima with erythrophagocytosis (arrows). 92
contralateral principal joint. The intima in groups 2, 3 and 4 was generally composed of 1 to 3 cell layers of synoviocytes and their subintima was essentially normal in cellularity.
Synovial Membrane-Transmission Electron Microscopy
The intima of control joints was typically composed of
1 to 3 cell layers of synoviocytes compatible with the type
A and B cel l s of Gha d i a l l y (or the type A and S cells respectively of Graabaek). Type A cells were usually more superficial and smaller than the subjacent type B cells
(Fig. 16). The normal type A cell was typically composed of: several long and slender cytoplasmic processes; several small cytoplasmic vacuoles; scant amounts of endoplasmic reticulum; few Golgi complexes; no surface caveolae; occasional coated vesicles and relatively large round mitochondria (Fig. 17). The normal type B cell was typically composed of: a few broad based cytoplasmic processes; abundant amounts of rough endoplasmic reticulum; many Golgi complexes; numerous surface caveolae; relatively small mitochondria; and no coated vesicles or cytoplasmic vacuoles (Fig, 17).
Morphologic changes in the principal synovial membranes were limited to group 1. The synovial intima was composed of 3 to 6 cell layers of synoviocytes (Fig, 18).
Type A synoviocytes were more abundant and larger, frequently exceeding the size of adjacent type B cells. In 93
Fig. 16. Normal control perimeniscal synovial membrane with smaller, more superficial type A cells (A) and larger subjacent type B cells (B). Bar = 2 um. 94
Fig. 17. A) Normal type A synoviocyte with several long cytoplasmic processes, scattered ribosomes, and large round mitochondria. Bar = .25 um. B) Normal type B synoviocyte with short blunt cell processes, surface caveolae, and abundant RER and Golgi complexes. Bar = .25 um. 95
Fig. 18. Group 1 perimeniscal synovial membrane from a principal joint. Four cell layers are obvious; type A cells (A) are large relative to the type B cells (B) and the superficial type A cells are markedly vacuolated. Bar = 2.5 u m . 96
type A cells lining the joint lumen, cytoplasmic vacuolation was markedly increased and tended to occupy the majority of the cytoplasm (Fig. 19). Vacuoles were large and some contained electron dense flocculent material (Fig.
19). Some vacuoles contained whorled membranous bodies and membrane-bound structures (Fig. 20). Cytoplasmic processes were more abundant, but still long and slender in type A cells. Type A cells positioned deeper in the intima were relatively large an4 also contained numerous cell processes, but lacked significant vacuolation.
The principal synovial membrane in groups 2, 3 and 4 were essentially normal.
Synovial Membrane-Scanning Electron Microscopy
The normal surface appearance of the synovial membrane varied within each joint, related to different anatomic sites. The typical perimeniscal synovial membrane had 1 to
3 large ridges adjacent and parallel to the articular surface of the meniscus (Fig. 21). These usually contained numerous villous projections and were covered by flattened cell bodies and tangles of cell processes. Peripheral to the perimeniscal ridges, the synovial membrane was relatively flattened and composed of gentle folds and furrows. These areas were covered by flattened cell bodies and tangles of cell processes. Small microplicae (surface folds) were present both on the cell bodies and cell processes. Collagen fibers were frequently seen in gaps 97
Fig. 19. A) Vacuolated type A synoviocyte containing many clear vacuoles. Bar = 2 um. B) Cytoplasmic vacuoles of type A synoviocyte containing flocculent material. Bar = 1 um. 98
Fig. 20. A) Type A synoviocyte with vacuoles containing whorled membranous bodies. Bar = 1 um. B) Enlargement of above showing vacuoles with whorled membranous bodies (W), flocculent material (F), and contracted membrane bound bodies (R). Bar = .25 um. 99
Fig. 21. Normal perimeniscal synovial membrane. A) The meniscus (M) is rimmed by ridges (R) of synovial membrane that eventually flattens (F) further out. Bar = 250 um. B) Synovial villi are either blunt or elongated. Bar = 100 um. C) Closer view of villi showing they are covered by flattened cell bodies and cell processes. Bar = 20 um. 100
between cell processes. In some areas, the membrane
abruptly changed from flattened, relatively hypocellular
areas to more cell rich areas covered by cells with rounded
cell bodies and thicker cell processes (Fig. 22).
The synovial membrane over the patellar fat pad was
poorly visualized and appeared glazed with a thick layer of
smooth material (Fig,23). Surface charging was a common
problem with the fat pad also. In some lipid-rich areas of
the perimeniscal synovial membrane , flattened synoviocytes
with long cell processes incompletely covered lipocytes in
the subintima.
Morphologic differences between principal and control
joints were limited to group 1. Large portions of the
perimeniscal synovial membranes were covered by densely
cellular areas. These areas were composed of closely
abutted, large, round cell bodies and tangles of cell
processes (Fig.24). Microplicae were numerous and
prominent.
Red blood cells were commonly seen in principal
joints from group 1. They were usually present in small
numbers scattered in various furrows and crevices of the
synovial membrane (Fig. 25). Some red cells were situated
between synoviocytes and synoviocyte processes extended
over them. Widely scattered, individual red cells were
rarely seen in principal joints. Large clumps of red cells were rarely seen on the cut surface of the synovium, 101
Fig. 22. A) Normal synovial membrane composed of both flattened synoviocytes (arrow) and rounded synoviocytes. Bar = 50 um. B) Flattened synoviocytes with numerous long cell processes and microplicae covering both processes and the cell body. Bar = 10 um. 102
Fig. 23. Typical appearance of synovial membrane from the patellar fat pad. Cells appear glazed by a heavy layer of material that obscures detail. Bar = 10 um. 103
Fig. 24. Perimeniscal synovial membrane from a group 1 principal joint. A) Hypercellular membrane composed of closely abutted large rounded synoviocytes. Bar = 5 um. B) Three synoviocytes with numerous large microplicae; synovial fluid droplet (S). Bar = 5 um. 104
Fig. 25. Red cells on the synovial membrane. A) Red cells were usually present in small numbers in synovial crevices and furrows. Bar = 10 um. B) Red cells interposed between synoviocytes with cell processes extending around them. Bar = 5 um. C) A large clump of red cells adhered to collagen fibers and lipid on the cut edge of a synovial membrane. Bar = 10 um. 105
adhered to collagen fibers and lipid droplets (Fig, 25).
These were interpreted as cells extravasated during the
collection procedure.
Meniscus and Skeletal Muscle
The menisci from the principal joints were essentially
the same as those from control joints. They were composed
of fibrocartilage containing prominent large collagen
fibers, roughly arranged parallel to one another.
Chondrocytes were frequently arranged in linear patterns
between the collagen fibers. Near the gliding surface,
chondrocytes were more dispersed in patterns similar to
hyaline cartilage.
There were no morphologic differences in the meniscal
surfaces between principal and control joints. The gliding
surface was relatively smooth and similar to the femoral
articular cartilage. The periphery of the meniscus was
abutted by synovial membrane. Numerous small parallel
folds of synovium that followed the curvature of the meniscus were consistently present.
Representative flexor (gastrocnemius) and extensor
(quadriceps femoris) skeletal muscles were examined by
light microscopy and found to be essentially normal in both
principal and control joints. 106
Biochemical Analyses of Articular Cartilage
Total Protein Assay
There was no significant difference between principal
and control joints in total protein concentration of
articular cartilage (Table 2)* Mean total protein
concentration was lower in the principal joints relative to
controls in groups 2 and 3 (Fig* 26), but this was not
statistically significant.
Hexosamine Assay
There was a significant difference (P = .019) between
the principal and control joints in the hexosamine content of articular cartilage (Table 3). There was a step-wise decrease in hexosamine concentration in groups 1, 2 and 3
(Fig. 27). However, this apparent trend was not statistically significant. In group 4, the hexosamine concentration was slightly greater than the counterpart controls.
Hydroxyproline Assay
There was no statistical difference between the principal and control joints hydroxyproline concentration of articular cartilage (Table 4) and (Fig.28),
Metabolic Analyses of Articular Cartilage
Articular Cartilage Production
There was no significant difference in cartilage total protein production between principal and control joints 3 7 5 □ Principal 350 TOTAL PROTEIN ASSAY ■ Control
o> 225
oi 150 3 '
Fig. 26. Results by group of the total protein assay articular cartilage.
100 HEXOSAMINE ASSAY H Control [ [Principal
3m 6m Time
Fig. 27, Results by group of the hexosamine assay of articular cartilage. riua cartilage. articular ug/mg Cartilage i. 8 Rsls y ru o te yrxpoie sa of assay hydroxyproline the of group by Results 28. Fig. YRXPOIE ASSAY HYDROXYPROLINE Principal □ Control I
108 109
(Table 5). There was also no significant difference in cartilage proteoglycan production between principal and control joints (Table 6). Both assays had a broad range of values due to differences among individuals, but values for counterpart joint pairs generally were similar.
There was a significant difference (P =.027) between principal and control joints for cartilage collagenous protein production (Table 7). Principal joints consistently averaged more than controls in all four groups.
Secreted Products
There was no significant difference in secreted protein production between principal and control joints
(Table 8). There was also no significant difference in secreted proteoglycan production (Table 9).
Morphometric Analyses
Synovial Membrane
The synovial membrane cellularity measured in cells per linear mm of membrane was significantly increased (P =
.037) in the principal joints over the control joints
(Table 10). This was primarily due to a nearly three-fold increase in cellularity in group 1 (Fig. 29). Cellularity in groups 2, 3 and 4 were similar between principal and control joints.
The hyperplastic synovial membrane reaction was also confirmed by another measurement; the thickness of the Synovial Cellularity □ Principal
350 r Control
« 1 0 0
7d 2m 3m 6m Time Fig, 29. Results of the morphometric analyses of synovial membrane, expressed in cells per linear mm of synovium.
Synovial Intimal Thickness
70 EH Principal 60 I Control 50
V> 40 « o c 30 X. o !e 20 H 10
2 m 3m 6 m Time
Fig. 30. Results of the morphometric analysis of synovi membrane expressed in um of thickness of the synovial membrane intima. Ill
synovial intima. There was a significant increase (P =
.038) in the intimal thickness of principal joints compared to control joints (Table 11). This also was primarily due to a marked increase in thickness in group 1 principal joints(Fig.30).
Articular Cartilage
The cellularity of articular cartilage was significantly increased (P = .01) in principal joints relative to control joints (Table 12). The differences were not obvious when examined by light microscopy except in rare cases where the difference was marked (Fig. 31),
The difference by groups was evident in group 1, greatest in group 2, and had subsided by group 4 (Fig. 32),
The average lacuna area of principal joints was not statistically different from the control joints (Table 13).
Group 1 principal lacunae approached being significantly larger (P = .058) than the controls (Fig. 33).
The matrix-cell area ratios (Table 14) and the percentage of articular cartilage occupied by lacunae
(Table 15) were not statistically different when comparing principal to control joints.
The subchondral bone area (Table 16) and the osteocyte lacunae area of subchondral bone (Table 17) were not statistically different when comparing principal and control joints. Fig. 31. Weight-bearing femoral articular cartilage from contralateral joints. A) Principal joint with 670 chondrocytes per mm . B) Control joint with 350 2 chondrocytes per mm1 1000 CELLULARITY | Control | | Principal
E 600
F 400
3m 6m Time
Fig. 32. Results of the morphometric analysi s o f articular cartilage, expressed in cells/mm of cartilage
150 r CHONDROCYTE LACUNAE AREA
^ Control
| | Principal
2 m 3m Time
Fig. 33, Results of the morphometric analysis of articular cartilage; average lacunae area measured in urn DISCUSSION
Morphologic Interpretation
Morphologic changes in acute hemarthrosis in rhesus monkeys are comparable to the natural and experimental disease of other species. Gross changes in this study were minimal. There were no gross changes in the articular surfaces and only mild patchy synovial membrane hemosiderosis. This is compatible with other hemarthrosis studies, for marked synovial membrane hemosiderosis has only been experimentally produced by repeated injections of
9 9 0 11? blood in rabbits and dogs. Yellowish discoloration of articular cartilage usually occurred, only after multiple bleeding episodes.*- However in horses, yellow discoloration accompanied chondromalacia, fibrillation and erosions following a single intra-
1 9 articular injection of blood.
The injected blood was not visible in principal joints by the time of necropsy with the exception of one monkey in the 7-day group, where a few small clots were found. Blood in joints of hemophiliacs and experimentally injected blood in rabbits is usually present as liquid blood with few, if any, small clots. In rabbits, blood was completely removed four days following experimental injection into the knee joint.217
R emoval of blood from the joint in this study was by
114 115
at least three routes. Erythrophagocytosis by mononuclear
cells was observed by light microscopy in both the intima
and subintima. In the intima this was interpreted as
synoviocyte erythrophagocytosis; but in the subintima,
these large mononuclear cells could have been either
synoviocytes that migrated into the subintima or
macrophages, Erythrophagocytosis by synoviocytes was
confirmed by transmission EM (Fig. 20), where
phagolysosomes contained contracted membrane-bound
structures and whorled membranous structures interpreted as
red cells and their membranes. The ability of synoviocytes 182 to phagocytize erythrocytes was first shown in rabbits
and occurred as rapidly as four hours post injection (PI).
At that point in time, phagocytized red cells were
recognizable and early degradation of the red cells
included microseptation by membranes of the phagolysosome.
Myelinoid bodies are osmiophilic membranes and when present
in synoviocytes are believed to be residues originating Q 1 from phagocytized erythrocyte membranes.
Blood removal in rhesus monkeys also occurred by the
incorporation of small clots into the synovium. Fibrin and
red cells were adhered to the synovial surface. Flattened
cells, which were contiguous with the synovial membrane,
formed an encompassing layer around some clots. There was marked synovial membrane hyperplasia at the point of adherence. 116
By scanning electron microscopy (EM), red cells were found in crevices of the synovial membrane, interposed between synoviocytes, suggesting exit of red cells by migration through the intima. The ability of red cells to change shape during diapedesis is well known. Similarly, red cells have been shown by transmission EM in previous hemarthrosis studies to migrate through the intima between 01 n synoviocytes. 7 In this study, a proliferative and inflammatory reaction in the synovial membrane was microscopically evident at 7-days PI but had subsided by 2- 10/ months PI. Key followed the synovial membrane reaction of rabbits after a single injection of homologous blood.
He found the inflammatory reaction to begin after one day
PI, peak at three to four days PI, and subside by seven days PI. Synoviocyte hypertrophy and hyperplasia in rabbits peaked at five days PI and regressed by 12 days PI.
0 1 Q Roy and Ghadially 7 reported morphologic changes in traumatic hemarthrosis of humans . They reported minimal changes at one to three days PI but focal proliferative changes at four days PI. Two weeks after traumatic hemarthrosis, patchy proliferative changes and mild inflammatory changes were still present in humans. These observations from the human study and our study in rhesus monkeys suggests that the general reaction to hemarthrosis in primates persists longer than that in rabbits.
Species variability in the synovial membrane reaction 117 to hemarthrosis is further supported by the work of 12 Ayers, who utilized horses. Ayers found the synovial membrane reaction following a single injection of autogenous blood to peak at 15 days to one month after injection. The reaction was characterized by synovial membrane hyperplasia and a mixed inflammatory cell reaction.
Therefore, the general reactions of synovial membranes to acute hemarthrosis by different species can be summarized as follows: a) the initial proliferative and inflammatory reaction begins early in all species studied
(between 1-3 days), b) and the reaction persists longest in the horse (15-30 days), intermediate in man and rhesus monkeys (7-14 days), and shortest in rabbits (4-5 days).
U1trastructural differences between control and principal synoviocytes were apparent in Group 1. The phagocytic type A cell was particularly affected. Type A cells were more numerous and relatively larger than type B cells, indicating hyperplasia and hypertrophy. In control joints, type A cells were generally smaller and present in approximately equal numbers relative to type B cells. In other hemarthrosis studies, changes in cell populations vary or were undetermined. In chronic hemarthrosis, synoviocytes usually contain numerous siderosomes making Q 1 the cell type unidentifiable. In human traumatic hemarthrosis, Roy and Ghadially reported an increase in 118
n i q type B and intermediate synoviocytes. In acute experimental hemarthrosis in rabbits, the same authors did not state the predominant cell type, however their figures show erythrophagocytosis by cells with numerous long filopodia and many cytoplasmic vacuoles that are most 7 1 7 compatible with type A cells. The cytoplasmic vacuoles of type A cel l s are considered to be endocytotic in n a t u r e ^ ’^® and as in this study were frequently increased in size and number when stimulated to absorb material from the joint space.-*-*^*^ jn rhesus monkeys, vacuoles frequently contained flocculent material identical to that found in the joint space, interpreted to be endocytosed joint proteins. Whorled membranous bodies interpreted as o] 917 9 1 Q residual erythrocytic membranes by others, were also found in this study. Long cytoplasmic processes
(filopodia) were prominent in the same reactive type A synoviocytes. The apparent scanning EM counterpart to filopodia are numerous large microplicae. Roy and 217 Ghadially showed filopodia extending around erythrocytes as an apparent preliminary event to incorporating the red cell into its cytoplasm as a heterophagosome. Although filopodia holding red cells was not observed by transmission EM in this study, scanning EM did show cell processes partially surrounding red cells. Hyperplasia and hypertrophy were also suggested by scanning EM, for the cells were more rounded and closely abutted, compared to 119 the flatter and more loosely arranged surface appearance of control joints.
The scanning EM appearance of the normal patellar fat pad synovial membrane was probably artifactual1y produced.
Unlike the perimeniscal synovial membrane, cells and cellular detail were obscured by a thick layer of material, giving it a 'glazed' appearance. This type of appearance has been demonstrated previously, especially in synovial membranes rich in l i p i d . >218,282,285 iower magnifications, a cobblestone pattern is apparent and is due to underlying lipid droplets. The glazed appearance may be due to lipids leaking from lipid droplets and coating the cells, or due to a thick coating of synovial fluid. However, studies on the behavior of synovial fluid on joint surfaces showed synovial fluid to appear as irregular and cracked thin sheets of material, or as multiple variably sized droplets. 0 In support of this material being of lipid origin in this study, was the observation that even after complete processing and dehydration, lipid would exude from the tissue specimen when gently squeezed.
U1trastructura1 changes in articular cartilage were mild and limited to superficial chondrocytes in group 1 principal joints. Membrane-bound cytoplasmic vacuoles found in some chondrocytes (Fig. 8) were usually peripherally located in the cell and frequently contained 120 material identical to that in the pericellular matrix.
These peripheral vacuoles appeared to be formed by endocytosis of the pericellular matrix via long cytoplasmic processes. In a few cells, vacuoles tended to coalesce and vacuole boundaries were indistinct. Numerous cytoplasmic processes of chondrocytes have been previously described in hemarthrotic cartilage of humans and dogs.'*''*'^ Increased length and number of cytoplasmic processes and increased cytoplasmic vacuolation in chondrocytes may be a morphologic indication of increased endocytotic turnover of the pericellular matrix. Similar changes have been described in rabbits with experimental 1ipoarthrosis, where cytoplasmic processes were shown to wrap around pericellular lipid droplets and incorporate them into the cytoplasm.
Another change present in the superficial group 1 chondrocytes were increased number of residual bodies and myelin figures relative to controls. Myelin figures have been reported previously in natural and experimental t c n 1 c hemarthrosis ’ and were interpreted to be foci of cytoplasmic degeneration (autophagolysosomes) that developed secondary to the noxious effects of intra- articular hemorrhage. Myelin figures have also been produced in cultured chondrocytes exposed to hemoglobin and ^ Q FeSO^. The component in blood most likely responsible for chondrocyte degeneration is ionic iron. In a study of 121 human hemophilic cartilage, chondrocyte vacuolation and necrosis was especially common in iron-containing cells as 9 C Q verified by microprobe analysis. Cytotoxicity of iron was manifested by a dose-dependent reduction in cell O Q numbers in cultured chondrocytes exposed to FeSo^. In this study, siderosomes were rarely seen as would be expected from a single intra-articular injection of blood.
Consequently, morphologic changes in chondrocytes were mild.
The reason for failure to localize enzymes ultrastructurally in cartilage and synovium was not apparent. The procedure used requires active enzymes to react with the substrate. It is possible that the prolonged fixation and travel time prior to processing inactivated the enzymes. An attempt to avoid this problem by transferring the tissues to buffer after 45 minutes of fixation, while enroute to the laboratory, still produced negative results. Another factor to consider is the thickness of the tissue. It is recommended that enzyme localization be performed with sections 50 urn thick or less so that the incubation media adequately penetrates the 919 tissue. Sections were diced as small as possible with razors, and care was taken to only examine the outer 50 um of tissue. This should have allowed the outer margins of the tissue to react with the substrate. 122
Biochemical, Metabolic and Morphometric Studies
Biochemical analysis revealed a significant decrease overall in hexosamine (proteoglycan) concentration in the principal joints. There was a progressive decrease in proteoglycan content in the first three groups and a return to normal by six months post injection. However, statistically this downward trend was not significant.
Decreased proteoglycan concentration was previously noted in experimental hemarthrosis in dogs.^ In that study, following multiple injections of autologous blood, glycosaminoglycans decreased progressively with time.
However, unlike this study which used only a single blood injection, their multiple injection study also showed a decreased collagen concentration in the later groups.
Biochemical analyses have not been reported in other hemarthrosis studies.
Decreased Safranin-0 affinity in articular cartilage has generally been considered a histochemica1 indicator of decreased proteoglycan concentration. The validity of
Safranin-0 as a histochemica1 indicator of proteoglycan in this study was verified by biochemical analysis of the articular cartilage. The observation that decreased affinity was greatest in the superficial zones of the articular cartilage suggests a noxious stimulus from the joint space.
Decreased proteoglycan content is a hallmark 123 biochemical change in osteoarthritic cartilage.5,33,1^3,161’^69 The decreased proteoglycan concentration is sometimes accompanied by an increased rate of proteoglycan synthesis; an apparent regenerative attempt to replace the lost proteoglycans.^4,227,264,266 Although there was a significant decrease in proteoglycan concentration in this study, there was no increase in proteoglycan synthesis. The reason for this may have been that the insult or decrease was not severe or prolonged enough for a metabolic response from the chondrocytes. One study found increased proteoglycan synthesis only to occur in the earlier stages of osteoarthritis and to decrease in
9 f i f i 9 f il l. the later stages, Teshima et al found increased
"^So^ incorporation only in cell suspension culture systems derived from osteoarthritic human cartilage that graded between 4 and 8 on the Mankin scoring system. The average
Mankin score of 6.9 for principal joints in this study falls within the range where an increase in proteoglycan synthesis might be expected. However, direct comparisons are not possible because this study utilized an organ culture system versus the cell suspension culture system used in their study, as well as species differences. Some studies have shown proteoglycan synthesis to be independent of histopathologic grading systems for osteoarthritic cartilage.9 5 ’163
There was a significant increase overall in 124 collagenous protein production in the principal joints.
This seems to contradict previous rationale that the insult in this study was not severe enough to produce a metabolic stimulus to chondrocytes (in regards to proteoglycan synthesis). However, the metabolic pathways and controls for the production of these two major matrix components are distinct as exemplified by their marked differences in
0 7 metabolic turnover. Increased collagenous protein production has been frequently reported in osteoarthritic cartilage.63>64» 132,144,180,202 In thig study) it is interpreted as a response to the intra-articular injection of autologous blood.
There was an overall significant increase in cellularity of articular cartilage from principal joints.
By groups, the principal joints consisitently averaged more than the controls; but a significant difference (when broken into groups) was only apparent in group 2. An increase in articular cellularity has not been shown previously in hemarthrosis studies, but has never been evaluated by morphometric methods. The cellularity differences in this study would not have been apparent by routine light microscopic observations, except in a few cases where differences were marked. In a hemarthrotic study using immature dogs, there was a significant increase in the thickness of articular cartilage following multiple 112 injections of blood. Morphometric analysis was not done 125 in that study, but the matrix-cell ratio was interpreted as normal and the change in thickness was termed 'cartilage overgrowth'. The ability of adult articular cartilage to respond to stimuli by undergoing division is probably best exemplified by chondrocyte clones that form along clefts and beneath fibrillated cartilage. Division in these cases is an apparent regenerative attempt by the articular cartilage to repair the architectural defect. However, chondrocyte clone formation has also been reported in osteoarthritic cartilage where the overlying surface architecture was normal.Verification of chondrocyte replication metabolical ly has been confirmed in osteoarthritic human hips using H-thymidine uptake studies.That study showed increased ^H-thymidine uptake by chondrocytes, but overall stable concentrations of DNA in the cartilage; this indicated chondrocytes were being lost and replaced in equilibrium. To further verify the morphometric indication of cartilage hypercel1 ularity in this hemarthrosis study, H-thymidine uptake studies and measurement of DNA levels would be necessary.
Hemarthrosis and CPPD Crystals in Rhesus Monkeys
The hypothesis that an intra-articular injection of autologous blood in rhesus monkeys could produce CPPD crystals in articular cartilage was not confirmed in 126
this study. The rationale for the hypothesis was based on
the fact that synovial membrane hemosiderosis was a common
finding in naturally occurring CPPD-DD in rhesus ono monkeys. Two mechanisms for crystal production were
possible in this study. The first is that endogenous
pyrophosphatase is strongly inhibited by divalent metal cations such as iron.^ ’ Failure to breakdown PPi
by pyrophosphatase could lead to accumulations in tissues at levels compatible with crystal precipitation. Support
for this mechanism is strong for the disease hemochromatosis, in which iron is deposited in large
amounts in multiple tissues including synoviocytes and chondrocytes. CPPD crystals were reported in over 50% of hemochromatosis patients with arthropathy.^^ Further support comes from reported cases of CPPD crystals in human hemophiliacs.'*-19,255 chondrocyte hemosiderosis was rare in this study and limited to superficial cartilage zones.
Although mild degenerative changes were present morphologically, the rarity of hemosiderin within chondrocytes suggests that a single injection of autologous blood may have been insufficient to test this hypothesized mechanism in rhesus monkeys.
The second possible mechanism for crystal production in this study is that the noxious stimulus of hemarthrosis could have produced a general increase in metabolic activity of articular cartilage. This occurs commonly in 127
degenerative joint disease. Support for this mechanism is
found in primary hyperparathyroidism, which is often 90S associated with CPPD-DD in humans. The mechanism is
believed to be related to increased calcium levels in
tissues as well as hormone induced increased metabolic o 9 activity and PPi production. Another factor to consider
in the monkeys from this study is their older age. It is
well recognized that variation of chemical composition with
age is common in humans. 2 7 9 This is believed to be related
to metabolic changes that occur with ageing. In rabbits
for example, there is a two fold increase in metabolic
activity (expressed on a per cell basis) in cartilage from
young versus old rabbits. 2 S fS This suggests that younger
individuals may be more responsive to metabolic stimuli
than older individuals. Although there was a significant
increase overall in collagenous protein production in this
study, crystals were not produced.
If crystal production was successful, it was
anticipated that small crystals would form in the
pericellular and territorial matrix of the transitional and
radial zones. Small crystals were occasionally seen in
those areas in both principal and control joints. Some
crystals were polyhedral and moderately electron dense; morphologically compatible with CPPD crystals (Fig. 12).
However, these crystals were too small to produce an x-ray
diffraction pattern for definitive identification. 128
Why significant crystal production was not produced
can only be speculated upon.. A possible reason is that the
insult produced by a single injection of autologous blood was not severe or prolonged enough for the hypothesized mechanisms to act. Perhaps multiple injections over
prolonged periods would have produced different results.
It is also possible that other unknown factors in addition
to hemarthrosis were necessary to produce crystals. CONCLUSIONS
The conclusions of this hemarthrosis study related to
the experimental objectives follow:
Objective: To determine if massive acute hemarthrosis in rhesus monkeys could produce CPPD crystals in the matrix of articular cartilage.
Conclusion: There was no significant crystal formation in the pericellular or territorial matrix of femoral articular cartilage when comparing injected and control knees.
Perhaps the joint insult produced by hemarthrosis in this study was not prolonged or severe enough to allow hypothesized mechanisms to act. Support for this speculation is the observation that hemosiderin within chondrocytes was rarely observed; which suggests the potential noxious or stimulatory effects of iron on chondrocytes would be minimal. The older age of these monkeys may have been a factor in their ability to respond metabo1ical ly to the joint stimulus. It is also possible that other unknown factors, in addition to heraosiderosois may be necessary for the formation of CPPD crystals in rhesus monkeys.
Ob j ective: To study the effect and recovery from a single episode of massive acute hemarthrosis in rhesus monkeys and
129 130
to fill a void in the literature on experimental hemarthrosis in subhuman primates.
Conclusion: A single injection of autologous blood into knee joints of rhesus monkeys produced an acute hyperplastic and inflammatory reaction in the synovial membrane which was marked at 7-days post injection (PI); but had resolved by 2-months PI. The hyperplastic synovium was confirmed morphometrically by measuring synovial membrane cellularity and synovial intima thickness.
Morphologic changes observed by electron microscopy in synoviocytes included increased numbers, size, and cytoplasmic vacuolation of type A cells and prominent microplicae. Synovial membrane hemosiderosis was evident in most injected knees, but decreased in severity with time. The injected blood had been completely removed from three of four joints by 7-days PI. Significant changes in articular surfaces were not produced, although mild degenerative changes were noted in superficial chondrocytes from group 1; including cytoplasmic vacuolation, increased myelin figures and rare hemosiderin deposits. Biochemical and metabolic studies revealed an overall significant decrease in cartilage proteoglycan content and increased collagenous protein production of principal joints.
Morphometry of articular cartilage revealed an overall hypercellular change relative to controls. The general reaction of the synovial membrane in rhesus monkeys was 131
similar to the rabbit and dog, but peaked later and lasted
longer. It appears that rhesus monkeys are also similar to
dogs and rabbits in the cartilage changes produced by hemarthrosis, since only mild morphologic changes and early resolution were observed following a single hemarthrotic episode. Rhesus monkeys are unlike horses, which developed chondromalacia and severe arthropathy 3-months after a single hemarthrotic episode.
Ob iective: To study the initiating events leading to cartilage destruction in blood-induced arthropathy by adding biochemical, metabolic and morphometric data to the traditional morphologic observations.
Conclusion: Massive acute hemarthrosis in rhesus monkeys resulted in an overall significant decrease in proteoglycan concentration of articular cartilage in principal joints.
The difference between principal and control joints was progressively greater up to 3rmonths PI and resolved by 6- months PI. This was accompanied by mild degenerative changes in superficial chondrocytes. Further evidence that hemarthrosis produced a noxious stimulus to articular chondrocytes is that there was an overall significant increase in collagenous protein production and increased cartilage cellularity in principal joints. Therefore, this study suggests that the initial events in blood-induced cartilage destruction are related to the adverse influence 132 of autologous blood on chondrocytes and their inability to maintain the cartilage matrix. It is well recognized that persistent and severe loss of proteoglycans eventually produces loss of cartilage architectural integrity and destruction. BIBLIOGRAPHY
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Animal Mo. Group No. Control Joint Principal Joint
1 1 7 6 2 1 6 6 3 1 8 8 4 1 4 8 5 2 7 9 6 2 7 14 7 2 8 6 8 2 7 7 9 3 11 6 10 3 5 5 11 3 7 7 12 3 4 5 13 4 2 6 14 4 5 6 15 4 12 5 16 4 6 7
Mean + SD= 6.6 + 2.5 6.9 + 2.2
P(T-Pair) * = NSD
Group Analysis
Mean + SD
Group Mo. Control Joint Principal Joint P(T-Pair)** to to 1 . 1.7 7.0 + 1.2 NSD 1 + 1
2 7.2 + .50 9.0 +3.6 NSD
3 6.8 + 3.1 5.8 + 1.0 NSD
4 6.3 + 4.2 6.0 + .82 NSD
P(1-AN0VA)= NSDNSD
*n=16 r **n=4, NSD= No significant difference, SD = standard deviation. 158 TABLE 2: Cartilage Total Protein Assay (ug/mg)
Animal No. Group No. Control Joint Principal Joint
1 1 141.1 99.6 2 1 200.5 135.2 3 1 190.5 249.8 4 1 129.7 195.6 5 2 395.5 119.1 6 2 220.5 151.1 7 2 101 .9 261 .0 8 2 297.4 180.7 9 3 332.1 124.8 10 3 261 .4 291.7 11 3 155.8 208.9 12 3 156.2 110.2 13 4 178.1 133.2 14 4 255.8 237.2 15 4 192.3 227.1 16 4 121.3 166.9
Mean + SD= 208.1 + 8 1 . 7 180.7 + 59.8
P(T-Pair) * = NSD
______Group Analysis
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)**
164.4 + 35.2 170.0 + 66.3 N SD
253.8 + 124.0 177.9 + 60.8 NSD
226.3 + 86.2 183.9 + 84.0 NSD
186.9 + 55.2 191 .1 + 49.5 NSD
P(1-ANOVA)« NSD NSD
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. 159 TABLE 3: Cartilage Hexosamine Assay (ug/mg)
Animal Mo. Group No.______Control Joint Principal Joint
1 1 66.9 54.5 2 1 103.5 53.5 3 1 50.4 38.9 4 1 43.9 36.5 5 2 53.7 14.8 6 2 59.3 26.7 7 2 42.8 78.1 8 2 31 .0 21 .5 9 3 91.7 42.7 10 3 41 .9 10.0 1 1 3 43.9 5.0 12 3 57.8 28.9 13 4 17.2 15.8 14 4 83.8 75.5 15 4 38.4 72.9 16 4 24.1 37.1
Mean + SD= 53.1 + 23.7 38.2 +
P(T-Pair)* = .019
Group Analysis
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)*
1 66.1 + 26.7 45.8 + 9.5 NSD
2 46.7 + 12.5 35.3 + 28.9 NSD
3 58.8 + 23.0 21.6 + 17.4 .001
4 40.8 + 29.9 50.3 + 28.9 NSD
P(1-ANOVA)= NSD NSD
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. 160 TABLE 4: Cartilage Hydroxyproline Assay (ug/mg)
Animal No. Group No.______Control Joint Principal Joint
1 1 27.5 36.1 2 1 25.6 22.4 3 1 23.4 26.2 4 1 29.4 31 .4 5 2 17.4 25.5 6 2 25.7 36.0 7 2 31 .1 27.3 8 2 21.6 28.1 9 3 20.3 20.4 10 3 26.2 24.0 11 3 28.7 15.7 12 3 23.2 31 .4 13 4 18.4 30.1 14 4 20.1 24.7 15 4 27.8 21 .4 16 4 26.1 29.9
Mean + SD= 24.5 + 4.1 26.9 +
P(T-Pair)* - N S D
______Group Analysis______
Mean + S D
Group No. Control Joint Principal. Joint P(T-Pair)** t o C h 1 26.5 + . 29.0 + 5.9 NSD
2 23.9 + 5.8 29.2 + 4.6 NSD
3 24.6 + 3.6 22.8 + 6.6 NSD
4 23.1 + 4.5 26.5 + 4.2 NSD
P(1-ANOVA)= NSDNSD
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. 161 TABLE 5: Cartilage Total Protein Production (cpm/mg)
Animal No. Group No. Control Joint Principal Joint
1 1 66,305 55,063 2 1 61,315 65,358 3 1 28,881 39,126 4 1 72,806 28,900 5 2 20,909 29,973 6 2 25,073 23,482 7 2 17,328 16,247 8 2 15,819 26,134 9 3 41 ,916 38,451 10 3 2,281 20,042 11 3 537 3,410 12 3 68,057 58,003 13 4 75,819 58,576 14 4 97,830 92,340 15 4 41 ,819 83,180 16 4 83,114 75,229
Mean + SD= 44,988 + 30,435 44,595 + 25,87'
P{T-Pair) * = N S D
Group Analysis
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)**
1 57,326 + 19,538 47,111 + 16,244 NSD
2 19,782 + 4,122 23,959 + 5,790 NSD
3 28,197 + 32,729 29,976 + 23,535 NSD
4 74,645 + 23,722 77,331 + 14,325 NSD
P{1-ANOVA)= NSD NSD
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. TABLE 6: Cartilage Proteoglycan Production (cpm/mg)
Animal No. Group No. Control Joint Principal Joint
1 1 15,424 12,175 2 1 16,127 23,106 3 1 10,465 25,467 4 1 22,896 14,361 5 2 3,109 5,667 6 2 1 ,540 1 ,176 7 2 2,726 1,724 8 2 3,076 5,408 9 3 412 605 10 3 34 581 11 3 396 3,550 12 3 13,471 23,287 13 4 10,974 15,390 14 4 28,494 23,942 15 4 10,313 12,708 16 4 22,418 12,140
Mean + SD= 10,117 + 9,114 11,330 + 9,030
P(T-Pair) * = NS D
Group Analysis
Mean + SD
Group No. Control Joint Principal Joint P ( T - P a i r )**
1 16,228 + 5,109 18,777 + 6,495 NSD
2 2,612 + 735 3,493 + 2,373 NSD
3 3,578 + 6,597 7,005 + 1.0,943 NSD
4 18,049 + 8,908 16,045 + 5,452 NSD
P(1-ANOVA)=
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. TABLE 7: Cartilage Collagenous Protein Production (cpm/mg)
Animal No. Group No. Control Joint Principal Joint
1 1 256 286 2 1 87 286 3 1 190 175 4 1 30 159 5 2 110 34 6 2 180 326 7 2 130 142 8 2 86 106 9 3 281 264 10 3 27 135 11 3 4 10 12 3 268 178 13 4 293 247 14 4 356 401 15 4 201 320 16 4 105 303
Mean + SD= 162.7 + 106.6 210.7 + 110.5
P(T-Pair) * = .027
Group Analysis
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)**
1 140.7 + 101.4 226.5 + 69.0 NSD
2 126.5 + 39.9 152.0 + 124.4 NSD
3 145.0 + 149.9 146.7 + 105.7 NSD
4 238.7 + 109.5 317.7 + 63 NSD
P(1-ANOVA)= ------
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. 164 TABLE 8: Secreted Protein Production (cpm/mg)
Animal No. Group No.______Control Joint Principal Joint
1 1 43,044 33,153 2 1 34,974 33,287 3 1 12,456 10,233 4 1 20,322 13,105 5 2 9,087 4,583 6 2 8,073 7,820 7 2 4,157 5,534 8 2 5,705 8,960 9 3 23,475 3,975 10 3 7,520 10,287 11 3 1,499 2,400 12 3 35,589 23,688 13 4 24,256 21,876 14 4 40,666 53,196 15 4 17,730 25,818 16 4 35,241 26,443
Mean +_ SD= 20,237 + 14,056 17,772 + 14,108
P(T-Pair)* = NSD
______Group Analysis______
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)**
27,699 + 13,846 22,444 + 12,497 NSD
6,755 + 2,238 6,724 + 2,016 NSD
17,020 + 15,466 10,087 + 9,686 NSD
29,473 + 10,387 31,833 + 14,384 NSD
P(1-AN0VA)=
*n=16, **n=4, NSD=* No significant difference, SD = standard deviation. 165 TABLE 9: Secreted Proteoglycan Production (cpm/mg)
Animal No. Group No. Control Joint Principal Joint
1 1 2,526 1,994 2 1 2,452 2,315 3 1 2,055 2,060 4 1 2,439 1,552 5 2 342 169 6 2 283 256 7 2 268 300 8 2 272 376 9 3 117 759 10 3 140 241 11 3 950 1,230 12 3 2,573 1 ,855 13 4 949 1 ,230 14 4 2,008 3,510 15 4 721 1,115 16 4 1 ,147 923
Mean + SD= 1 ,202 + 969 1,242 + 935
P(T-Pair) * = N S D
Group Analysis
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)**
1 2,368 + 212 1 ,980 + 317 NSD
2 291 + 34 275 + 86 NSD
3 945 + 1 ,152 1 ,021 + 687 NSD
4 1 ,206 + 562 1 ,694 + 1 ,216 NSD
P{1-AN0VA)=
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. 166 TABLE 10: Synovial Membrane Cellularity fcells/mm)
Animal No. Group No. Control Joint Principal Joint
1 1 165 338 2 1 118 342 3 1 115 253 4 1 102 292 5 2 113 93 6 2 125 179 7 2 150 165 8 2 120 142 9 3 99 199 10 3 223 96 11 3 183 108 12 3 123 107 13 4 1 14 176 14 4 127 183 15 4 201 164 16 4 96 94
Mean + SD= 136.1 + 37.9 183.2 + 83.2
P(T-Pair) * =* .037
Group Analysis
Mean + SD
Group Ho. Control Joint Principal Joint P(T-Pair)**
1 125.1 + 27.1 306.2 + 41.9 .001
2 127.1 + 16.3 144.9 + 37.8 NSD
3 157.4 + 56.6 127.4 + 48.2 NSD
4 134.8 + 46.2 154.4 + 42 NSD
P (1 -A N O V A ) = NSD .038
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. TABLE 11: Thickness of Synovial Membrane Intima (um)
Animal No. Group No. Control Joint Principal J<
1 1 26 63 2 1 24 127 3 1 7 49 4 1 17 34 5 2 15 18 6 2 12 21 7 2 19 15 8 2 1 1 21 9 3 13 26 10 3 33 34 11 3 19 12 12 3 17 11 13 4 18 35 14 4 27 20 15 4 34 24 16 4 16 14
Mean + SD= 19.2 + 7.7 32.8 + 28
P(T-Pair) * = .038
______Group Analysis______
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)** 00 \£>
1 18.5 + • 68.3 + 40.1 .037
2 14.3 + 3.6 18.8 + 2.9 NSD
3 20.5 + 8.7 20.8 + 11.1 NSD
4 23.8 + 8.3 23.2 + 8.8 NSD
P (1-ANOVA)= NSD .01
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. TABLE 12: Articular Cartilage Cellularity (cells/mm2)
Animal No. Group No. Control Joint Principal Joint
1 1 522 536 2 1 328 343 3 1 438 572 4 1 352 406 5 2 285 361 6 2 394 578 7 2 350 671 8 2 462 490 9 3 394 362 10 3 271 526 11 3 268 390 12 3 533 522 13 4 267 438 14 4 519 413 15 4 256 292 16 4 427 355
Mean + SD= 397 + 97 453 + 106
P(T-Pair) * = .01
Group Analysis
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)**
1 410 + 88 464 + 108 NSD
2 373 + 75 525 + 132 .05
3 366 + 126 450 + 86 NSD
4 367 + 128 375 + 65 NSD
P(1-ANOVA)= NSD NSD
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. , 169 TABLE 13: Average Lacuna Area of Articular Cartilage (umz)
Animal No. Group No. Control Joint Principal Joint
1 1 70 63 2 1 96 163 3 1 57 93 4 1 79 117 5 2 72 91 6 2 46 55 7 2 59 55 8 2 45 50 9 3 74 87 10 3 65 93 11 3 40 53 12 3 91 88 13 4 49 71 14 4 88 22 15 4 70 98 16 4 60 49
Mean + SD= 78.0 + 33.3 66.3 16.9
P(T-Pair) * = NS D
Group Analysis
Mean + SD
Group No, Control Joint Principal Joint P(T-Pair)**
1 109 + 42.2 75.5 + 16.4 NSD
2 62.8 + 18.9 55.5 + 12.7 NSD
3 80.3 + 18.4 67.5 + 21.3 NSD
4 60,0 +_ 32.3 66.8 + 16.6 NSD
P(1-ANOVA)= NSD NSD
*n=16, **n=4, NSD= No significant difference, SD = standard deviation. 170 TABLE 14: Matrix Cell Area Ratios of Articular Cartilage
Animal No. Group No. Control Joint Principal Joint
1 1 29.4 25.5 2 1 17.6 29.3 3 1 23.6 29.6 4 1 23.2 30.2 5 2 29.4 37.8 6 2 44.8 36.6 7 2 50.7 24.1 8 2 41.9 44.0 9 3 30.9 33.2 10 3 38.8 28.2 11 3 69.8 63.3 12 3 20.3 20.0 13 4 51.5 45.6 14 4 38.5 26.4 15 4 39.1 47.9 16 4 46.9 45.7
Mean + SD= 37.3 + 13.8 35.4 + 1 1 . 3
P(T-Pair) * = NSD
Group Analysis
Mean + SD
Group No. Control Joint Principal Joint P(T-Pair)**
1 23.5 + 4.8 28.7 + 2.1 NSD
2 41.7 + 8.9 35.6 + 8.3 NSD
3 39.9 + 21.3 36.2 + 18.9 NSD
4 43.9 + 6.3 41.4 + 10.0 NSD
P (1-ANOVA)= NSD NSD
*n=16, **n=4, NSD- No significant difference, SD = standard deviation. TABLE 15: Percentage of Articular Cartilage Area Occupied by Lacunae (%)
Animal No. Group No. Control Joint Principal Joint
1 1 3.3 3.8 2 1 5.4 3.3 3 1 2.9 3.3 4 1 4.1 3.2 5 2 3.3 2.6 6 2 2.2 2.7 7 2 1.9 4.0 8 2 2.3 2.2 9 3 3.1 2.9 10 3 2.5 3.4 1 1 3 1.4 1.6 12 3 4.7 4.8 13 4 1.9 2.2 14 4 1 .2 3.6 15 4 2.5 2.1 16 4 2.1 2.1
Mean + SD= 2.8 + 1.2 3.0 + 1
P ______Group Analysis______ Mean + SD Group No. Control Joint Principal Joint P(T-Pair)** 1 3.9 + 1.1 3.4 + 0.26 N SD 2 2.4 + 0.59 2.9 + 0.77 NSD 3 2.9 + 1.4 3.2 + 1.3 NSD 4 1.9 + 0.56 2.5 + 0.76 NSD P(1-AN0VA)= NSD NSD *n=16, **n=4, NSD= No significant difference, SD = standard deviation. 172 TABLE 16: Subchondral Bone Area Expressed as Percentage of Total Subchondral Area (%) Animal No. Group No.______Control Joint Principal Joint 1 1 32.7 44.4 2 1 48.5 38.3 3 1 40.2 38.6 4 1 45.2 38.9 5 2 29.4 23.7 6 2 50.7 36.3 7 2 38.1 31.6 8 2 39.9 40.8 9 3 37.1 39.6 10 3 41.9 40.8 1 1 3 46.4 36.4 12 3 36.5 19.6 13 4 28.0 18.0 14 4 40.9 33.3 15 4 27.7 22.2 16 4 20.6 24.3 Mean + SD= 37.7 + 8.3 32.9 + 8.5 P(T-Pair)* = NSD Group Analysis______ Mean + SD Group No. Control Joint Principal Joint P(.T-Pair)** 00 1 41 .6 + * 40.0 + 2.9 NSD 2 39.5 + 8.7 33.1 + 7.3 NSD 3 40.5 + 4.6 34.1 + 9.8 NSD 4 29.3 + 8.5 24.4 + 6.4 NSD P {1-ANOVA)= NSD NSD *n=s16f **n=4, NSD= No significant difference, SD = standard deviation. TABLE 17: Osteocyte Lacuna Area of Subchondral Bone Expressed as Percent of Trabeculae Area (%) Animal No. Group No. Control Joint Principal Joint 1 1 1.7 1.9 2 1 1.3 1 .6 3 1 1.6 1.3 4 1 2.3 1.1 5 2 3.8 1.5 6 2 1.3 1.3 7 2 2.3 2.1 8 2 1.7 1.1 9 3 0.92 1 .3 10 3 1.1 1 .0 11 3 0.84 0.94 12 3 0.64 0.98 13 4 0.96 1.1 14 4 1 .3 1 .3 15 4 0.84 0.52 16 4 1.1 0.88 Mean +_ SD= 1.5 _+ .76 1.3 + .33 P(T-Pair)* = NSD ______Group Analysis______ Mean + SD Group No. Control Joint Principal Joint P(T-Pair)** 1 1.7 + .36 1.5 + .30 NSD 2 2.2 + .94 1 .5 + ,37 NSD 3 .87 + .17 1.0 + ,14 N SD 4 1.05 + .17 .95 + .29 NSD P(1-ANOVA)= NSD NSD *n=16, **n=4, NSD= No significant difference, SD = standard deviation. Curriculum Vitae Name: David Henry Zeman Birthdate: January 18, 1954 Birthplace: Coeur d1 Alene, Idaho Marital Status: Married , 2 children E d u c a t i o n : B.S, North Dakota State University Animal Science cum laude 1976 D.V.M. Oklahoma State University College of Veterinary Medicine 1980 Ph.D. Louisiana State University School of Veterinary Medicine Department of Veterinary Pathology Anticipated- May 1986 Doctoral Dissertation: Experimental hemarthrosis in rhesus monkeys: Gross, light and electron microscopic, biochemical, metabolic, and morphometric analyses. Specialty Boards: Board eligible November 1985 A. C. V. P. H o n o r s : Alpha Zeta 1975 Phi Kappa Phi 1976 B.S. cum laude, N.D.S.U. 1976 Charles L. Davis, D.V.M. Foundation, Veterinary Pathology Scholarship Award 1984 Gamma Sigma Delta 1985 174 175 Major Professional Interests: Food animal pathology Ultrastructural pathology Bone and joint pathology Wildlife and laboratory animal pathology Professional Experience: Graduate Teaching Assistant 1982-Present Louisiana State University School of Veterinary Medicine Department of Veterinary Pathology Duties: Participate in the departments diagnostic necropsy and biopsy service; assist in the instruction of 4th year vet. med. students in necropsy techniques and interpretation of gross pathologic lesions; conduct gross pathology labs of 2nd year vet. med. students(ro- tation); assist in the instruction of 2nd year vet. med. students in microscopic pathology labs; participate in departmental seminars and weekly AFIP histopatho 1ogy seminars. Delta Regional Primate Research Center 1983 Department of Pathology Covington,LA Experience: As a graduate assistant from L.S.U., I spent one day per week for approximately one year conducting necropsies on various species of monkeys under the direction of Dr. Gary Baskin of the primate center. Private Practice of Veterinary Medicine St. Cloud, Minnesota 1 9 8 0 - 1 9 8 2 Professional Organizations: American Veterinary Medical Association Minnesota Veterinary Medical Association Louisiana Electron Microscopy Society Ancillary Experience: Minor: Department of Veterinary Anatomy and Fine Structure, Electron Microscopy emphasis. Computer Skills: Have utilized the following IBM per sonal computer programs in my research: Wordstar, ABSTAT, and Bioquant Morphometries. 176 Publications: Encepha1itozoonosis in squirrel monkeys (Saimiri s c i u r e u s ). D.H. Zeman and G.B. Baskin. Vet Pathol 22:24- 31.1985. Lethal short-limbed dwarfism in a rhesus monkey. D.H. Zeman and G. B. Baskin. Vet Pathol 23:198-200,1986. Intracranial squamous cell carcinoma in a cow. D.H. Zeman and D.-Y. Cho. Cornell Vet (In Press) Ho 1 oprosencepha1 y in a bovine calf. D.-Y. Cho, D.H. Zeman and J.E. Miller. Acta Neuropathol (Ber 1) 67:322- 325.1985. Cerebellar Disease in an Adult Cow. H.H. Oz, S.S. Nicholoson, F.K. Al-Bagdadi and D.H. Zeman. Canadian Vet J 27:13-16,1986, Hemi1aminectomy using an autoclavable electric drill compared to a pneumatic drill. P.K. Shires, E.D. Roberts, D.A. Hulse, D.H. Zeman and M.T. Kearney, J Am An Hosp Assoc 22:25-29,1986. Abstracts: Cartilage metabolic studies in rhesus monkeys with pseudogout. E.D. Roberts, D.H. Zeman, G.B. Baskin, H. Shoje. Proceedings: 65th Conference of Research Workers in Animal Disease, 1984. Presentations: Encephalitozoonosis in squirrel monkeys: Neonatal and placental pathology. Reproductive Pathology Specialty Group Meeting. 35th Annual Meeting of the American College of Veterinary Pathologists, Toronto, Ontario, Canada, 1984. Experimental hemarthrosis in rhesus monkeys. Orthopedic Pathology Specialty Group Meeting. 36th Annual Meeting of the American College of Veterinary Pathologists, Denver, Colorado, 1985, Intracranial squamous cell carcinoma in a cow. The 13th Annual Southeastern Veterinary Pathology Conference, Tifton, Georgia,1985. 177 Presentations Continued: Granulomatous encephalitis due to E_j_ cunicul i in a squirrel monkey. The 12th Annual Southeastern Veterinary Pathology Conference, Tifton, Georgia, 1984. Endometriosis in a rhesus monkey. The 11th Annual Southeastern Veterinary Pathology Conference, Tifton, Georgia, 1983. Mailing Address and Phone Numbers: David H. Zeman, D.V.M, Department of Veterinary Pathology School of Veterinary Medicine Louisiana State University Baton Rouge, LA 70803 Office Phone: 504-346-;-3225 Home Phone: 504-923-3969 References: E. D. Roberts, D.V.M., Ph.D. Major Professor Louisiana State University School of Veterinary Medicine Department of Veterinary Pathology Baton Rouge, LA 70803 Phone: 504-346-3225 H. W. Casey, D.V.M., Ph.D. Professor and Department Head Louisiana State University School of Veterinary Medicine Department of Veterinary Pathology Baton Rouge, LA 70803 Phone: 504-346-3225 G. B. Baskin, D.V.M. Head- Department of Pathology and Adjunct Professor Delta Regional Primate Research Center 3 Rivers Road Covington, LA 70433 Phone: 504-892-2040 DOCTORAL EXAMINATION AND DISSERTATION REPORT Candidate: David Henry Zeman Major Field: Veterinary Medical Sciences; Option: Veterinary Pathology Title of Dissertation: Experimental Hemarthrosis in Rhesus Monkeys: Light Microscopic, Scanning and Transmission Electron Microscopic, Biochemical, Metabolic, and Morphometric Analyses. Approved: AIL.Dean of the Graduatlluate School EXAMINING COMMITTEE: ■ ( Date of Examination: April 29, 1986