Professional Documents
Culture Documents
THESIS
Submitted for the Partial Fulfillment of the Master Degree
"M.Sc." in Anatomy
Presented by
Supervised by
Faculty of Medicine
Ain Shams University
2004
ACKNOWLEDGEMENT
I am deeply indebted to Professor. Dr. Mostafa Kamel Ibrahim El-sayed (Professor of
Anatomy, Head of Anatomy Department, Faculty of Medicine, Ain Shams University) for
suggesting, planning and supervising this work, for providing all the laboratory facilities and
for his continuous guidance and encouragement.
I would like to express my deepest thanks and gratitude to Professor. Dr. Fouad Yehia
Ahmed and Assistant Professor. Dr. Hassan Mostafa Serry (Anatomy Department, Faculty of
Medicine, Ain Shams University), for their wise guidance, criticism and valuable suggestions
throughout the present study.
I would like to acknowledge Professor. Dr. Mohammed Fawzi GabAllah (Professor of
Anatomy, Faculty of Medicine, Cairo University) for his invaluable advice and suggestions.
Table of Contents
MORPHOLOGICAL STUDY OF THE MUSCLE- BONE INTERFACE IN MAN" ..... 1
ACKNOWLEDGEMENT ......................................................................................................... 2
INTRODUCTION ..................................................................................................................... 4
I-Macroscopic Features of Muscle-Bone Interface: .............................................................. 5
II-Microscopic Features of Muscle-Bone Interface: .............................................................. 6
III-Chemistry of Muscle-Bone Interface: .............................................................................. 8
IV-Structural Functional Correlation of Muscle-Bone Interface: .......................................... 9
V-Clinical Correlation of Muscle-Bone Interface: .............................................................. 12
VI-Tidemark in Ligament Insertions and Articular Cartilage: ............................................ 13
VII-Comparative Anatomy of Muscle-Bone Interface: ....................................................... 14
Material and Methods .............................................................................................................. 15
I- Choice of Muscle-Bone Interface Specimens: ................................................................. 15
II- Preparation of Muscle-Bone Interface Specimens for the Light Microscopic Study: .... 15
Results ...................................................................................................................................... 16
I- Tendon-Bony Prominences Attachment (Enthesis): ........................................................ 16
A) Enthesis of Biceps Brachii Muscle: ............................................................................ 16
B) Enthesis of Tendocalcaneus: ....................................................................................... 17
II- Examples of Linear Muscle-Bone Interface: .................................................................. 18
A) The External Intercostal Muscle: ................................................................................ 18
B) Brachioradialis Muscle: .............................................................................................. 18
C) The External Oblique Muscle: .................................................................................... 19
III- Examples of Broad Muscle-Bone Interface: ................................................................. 19
A) Infraspinatus Muscle:.................................................................................................. 19
B) Brachialis Muscle: ...................................................................................................... 20
Discussion ................................................................................................................................ 20
I- Tendon-Bony Prominences Attachment (Enthesis): ....................................................... 20
II- The Muscle-Bone Interface of Fleshy Linear Muscle Attachment:................................ 23
III- The Muscle-Bone Interface of Muscles Attached by Fleshy Fibers Over a Wide Bony
Area: ..................................................................................................................................... 24
IV) Bone Periosteum in Relation to Enthesis and Fleshy Muscle-Bone Interface: ............ 24
CONCLUSION ........................................................................................................................ 25
Figures...................................................................................................................................... 26
SUMMARY ............................................................................................................................. 42
References ................................................................................................................................ 43
Arabic Summary ...................................................................................................................... 47
INTRODUCTION
Each skeletal muscle has at least two attachments one at each end. These attachments
might be purely tendinous, fleshy, or an admixture of flesh and tendon. The pure tendinous,
always leave a smooth mark on the bone, the fleshy ones generally leave no mark on the bone,
while the rough marks are made where there is an admixture of flesh and tendon (Last, 1999).
Most tendons present four histological zones at their bony attachments: dense fibrous tissue,
uncalcified fibrocartilage, calcified fibrocartilage, and bone (Francois et al., 2001; Benjamin
and Ralphs, 2001). The presence of the uncalcified fibrocartilage offers some protection from
wear and tear, while the calcified fibrocartilage anchors the tendon to the bone and enables it
to withstand shear so that traumatic avulsion of tendon insertion rarely occur at the actual
interface with bone (Clark and Stechschulte, 1998).
The muscle-bone interface shows considerable regional heterogenicity in different
tendons that should be taken in consideration for selecting tendons for particular surgical
transfers or joint reconstruction (Benjamin et al., 1995). Orthopedic surgeons may need to
reattach damaged tendons and ligaments to bone, or to re-route tendons in treating injuries of
peripheral nerves. A successful union will best occur if the bone can grow into the
tendon/ligament and establish an enthesis that closely resembles the original (Rodeo, 2001).
Rheumatologists call tendon/ligament attachment zones (enthesis) and much current
interest is focused on their involvement in a group of conditions known as the (seronegative
spondyloarthropathies). The best known of these is ankylosing spondylitis in which individual
bones fuse together across joints (Benjamin and McGonagle, 2001).
Muscles and ligaments are common sites of both overuse and traumatic injuries in
sport, and the enthesis is one of the regions most commonly affected. Thus, tennis elbow for
example specifically affects the attachment of one or more tendons, which belong to muscles
that lie in the back of the forearm (Benjamin et al., 2002).
Reviewing the literature, a lot of work dealt with the structure of tendonbone
interface (enthesis). However, up to our knowledge, no comparable study was done on the
fleshy-bone attachment; where no apparent tendon is found and a broad or long fleshy-bone
contact is present. Taking into consideration that such form of muscle attachment might be
equally important as tendons in being the site of active movements, traumatic injuries, or
rheumatic disorders.
Therefore, it became the aim of the present study to investigate the histological
structure of the fleshy muscle-bone interface in selected limb muscles in man, as compared to
that of the enthesis, in an attempt to clarify the way muscle fibers transmit their contractile
force to adjacent bone, and to make use of it in the clinical practice.
Gao et al. (1994) mentioned that tendon fibrocartilage had oval or round cells
embedded in a highly metachromatic matrix with interwoven or spiraling collagen fibers. The
fibrocartilage cells were arranged in rows between parallel collagen fibers.
Gao and Messner (1996) conducted a histological quantitative comparison study on
the soft tissue-bone interface of femoral insertion of medial collateral ligament, both insertions
of cruciate ligaments, and the tibial insertion of patellar ligament in the rabbit. It was noticed
that at chondral ligament insertions, the calcified fibrocartilage interdigitated deeply with the
lamellar bone. Moreover, the authors found that the numbers and frequency of interdigitations
were lowest at the medial collateral ligament insertion. On the other hand, the medial collateral
ligament had the thickest zone of calcified fibrocartilage. The authors postulated that the
thickness of fibrocartilage might be more related to the amount of movement occurring at an
insertion.
Rufai et al. (1996) described the ultrastructure of three fibrocartilages at the insertion
of the adult rat Achilles tendon; (1) Enthesial fibrocartilage at the tendon-bone junction, (2)
Sesamoid fibrocartilage in the deep surface of the tendon and (3) Periosteal fibrocartilage
covers the opposing surface of the bone. Extracellular matrix was fibrous with little
proteoglycan, while the cells had rough endoplasmic reticulum, glycogen, and lipid, whereas,
pericellular matrix rich in proteoglycans and fine collagen fibrils. The periosteal fibrocartilage
developed as a secondary cartilage from the periosteum while enthesial and sesamoid
fibrocartilages developed by metaplasia of the tendon fibroblasts. A major difference between
the three fibrocartilages was the arrangement of their collagen fibrils. There were parallel
bundles in enthesial fibrocartilage but interweaving networks in the sesamoid and periosteal
fibrocartilages.
Raspanti et al. (1996) investigated the tibial insertion of the patellar ligament of the
rat by light microscopy, scanning electron microscopy and transmission electron microscopy.
The authors noticed that until the point of insertion, the patellar ligament showed the typical
structure of a tendon. However, in proximity to the insertion, the ligament was gradually
infiltrated by a different, cartilage-like matrix and the tenocytes became progressively rounded
and displayed some characteristics of chondrocytes. Then tendon fibers crossed this
fibrocartilage and appeared to interweave with the tibial bone.
Clark and Stechschulte (1998) reported that traumatic avulsions of ligament or tendon
insertions rarely occurred at the actual interface with bone, which suggests that this attachment
is strong or otherwise protected from injury by the structure of the insertion complex. The
authors studied quadriceps tendon fibers where they insert into the patellae of adult rabbits,
7
humans, dogs and sheep. Specimens were examined by scanning electron microscopy (SEM)
and light microscopy (LM). By SEM, it was possible to identify mature bone by the presence
of osteocytes and a lamellar organization. LM and SEM showed that, unlike tendon fibers
elsewhere, those in the calcified fibrocartilage were not wavy. Moreover, SEM identified no
specific cement line.
Trischer et al. (2002) studied the quadriceps tendon of the rabbit and reported the
presence of variety of proteoglycans (aggrecan and versican), glycosaminoglycans
(chondroitin-4 and -6 sulfate, dermatan sulfate, keratin sulfate), and glycoprotein (tenascin) in
its extracellular matrix (ECM) and vimentin in the fibrocartilage cells. The authors added that
the presence of aggrecan enables the tendon to withstand compression.
Tillmann et al. (1992) found that where more movement is permitted at the soft/hard
tissue interface, there is more uncalcified fibrocartilage, and where more force is transmitted
to the bone, there is a thicker layer of cortical calcified tissue and a greater proportion of bone
to marrow. The authors have found that there are pronounced differences in the quantities of
uncalcified fibrocartilage in different tendons and between the superficial and deep parts of
each enthesis. Rufai et al. (1992) mentioned that the functional significance of periosteal
fibrocartilage was to serve along with the associated tendon fibrocartilage to prevent tendons
and their pulleys from being damaged by the sawing action of the tendon. Vogel et al. (1993)
found that tibialis posterior is fibrocartilaginous where it passes around the medial malleolus.
Robbins and Vogel (1994) reported that the fibrocartilage enables the tendons to resist
compression because it contains large proteoglycans typical of cartilage. Weiss et al. (1994)
reported that pulley tissue was responsive to mechanical demands, for periosteal fibrocartilage
lining a bony groove might disappear when the associated tendon was ruptured and
fibrocartilage (as indicated by increased quantities of type II collagen) might appear in the
flexor retinaculum in patients with the carpal tunnel syndrome.
Benjamin et al. (1995) found that tendons attached to the tarsus and metatarsus had
fibrocartilaginous enthesis, but those attached to phalanges had fibrous enthesis. The authors
related these differences to variations in the movement of such tendons near their attachment,
and they concluded that the more mobile tendons had more fibrocartilage. Such enthesis
fibrocartilage had a mechanical role in preventing tendon fibers from fraying at bone
attachments. Benjamin and Ralphs (1995) mentioned that significant differences occurred in
the distribution of fibrocartilage between the upper and lower limbs. Fibrocartilage
differentiation was much more pronounced in tendons at the ankle than the wrist. This was
attributed to anatomical factors that resulted in mechanical differences. Because the long axis
of the foot is at right angles to that of the leg, tendons at the ankle were permanently bent
around the bony malleoli and thus constantly subjected to compression and/or shear. However,
in the wrist, there is little or no change in tendon direction when the hand is in the anatomical
position.
Frowen and Benjamin (1995) studied the relationship between the presence or amount
of fibrocartilage at the attachments of the major extrinsic muscles in the foot, and the extent to
which these tendons bent near their enthesis during movement. The authors concluded that
fibrocartilage at enthesis (tendon-bone junctions) prevented collagen fibers bending at the hard
tissue interface. Ralphs et al. (1995) described that the extensor tendons of the fingers are
similarly modified where they cross the proximal interphalangeal joints. Salmons (1995)
11
12
tidemark line. Middle-grade tidemark changes were characterized by vascular invasion into the
tidemark as well as incipient calcification of basal hyaline cartilage. High-grade tidemark
changes were characterized by the disappearance of the tidemark, advanced mineralization and
ossification of the basal hyaline and calcified cartilage.
Redler et al. (1975) examined the tidemark of human articular cartilage by scanning
electron microscopy and identified three bands: the first band consisted of randomly oriented
compacted fibrils that appeared to be continuous with those of the non-calcified and calcified
zones. The second band was formed of flattened fibrils paralleling the undulating surface of
the calcified cartilage. Finally, the third band comprised perpendicularly oriented fibrils having
a distinct continuous transition between the non-calcified and calcified zones.
Havelka et al. (1984) ascribed the tidemark as an interface, which might better be
defined by biomechanical methods than by morphology. It originates, by chondrocyte activity,
between calcified and non-calcified cartilage layers of any kind, hyaline or fibrous, in areas
exposed to either loading (joint) or pulling (insertion). In the articular cartilage it appeared with
skeletal maturation, in other localizations it was age-independent. It should be regarded as a
special instance of a broader phenomenon of the calcification/mineralization front. Inside the
joint cartilage, its changes reflected the slow remodeling of the calcified layer and its
unapparent shift towards the surface of the articular cartilage. In the marginal transitional zone
of the joint, tidemark smoothly passed into the periosteum. Chondrocytes on both sides of the
tidemark are positive for alkaline phosphatase and the positive reaction continuously goes on
to the periosteum.
Sagarriga et al. (1996) studied the collagen fibrils of fibrocartilages of the bovine
medial collateral ligament attachments to bone, and found that they attach to bone by passing
through a zone that consists of non-mineralized and mineralized fibrocartilages; type I, II, V,
IX and XI collagen were found. Especially type II and IX found in non-mineralized (mainly)
and mineralized zone of the insertion. The cartilage collagens play a role of anchoring the
ligament to bone or the cartilage-like tissue participate in the modulation of the mechanical
stresses which exist at the soft tissue-hard tissue interface.
Involvement of the periosteum in the fibrocartilage, (3) The presence of various types of
fibrocartilage cells in the tendon near the interface , to reinforce the tendon against compression
or shear stress, and (4) both fibrocartilage and hyaline cartilage (lateral articular cartilage)
receiving the tendon at the epiphyses. Overall, variations in reptilian bone-tendon interface
represent adaptations to the continuous growth and loose joint structures of lizards.
Suzuki et al. (2003) studied bone-tendon and bone-ligament interfaces in crocodile
limbs under light microscopy. The authors found that crocodilian interfaces included a direct,
unmediated insertion in which the tendon or ligament fibers inserted directly into the bone itself
without fibrocartilaginous mediation.
II- Preparation of Muscle-Bone Interface Specimens for the Light Microscopic Study:
In every case, the muscle was dissected and its attachment to the bone was exposed.
With the aid of chisel and hammer as well as the use of bone nibbling forceps, the whole
muscle-bone interface was extracted so that each specimen included the muscle and the
underlying bone tissues. The specimens were fixed in 10% neutral buffered formol saline for
one week, and then decalcified with 10% EDTA for about 4-6 weeks (Gao and Messner, 1996).
15
Dehydrated in ascending grades of alcohols, cleared in xylol, and embedded in paraffin wax.
In all paraffin blocks, the specimens were oriented so that the sections were cut to include both
soft tissue and bone. Serial sections were cut at 8-m thickness on a leitz rotatory microtome.
Staining with haematoxylin and eosin and Masson's trichrome was carried out (Drury and
Wallington, 1980).
Results
Since a good amount of work have dealt with the tendon-bone attachment and its
histological structure. Therefore, it was logic to start with its examination and take it as a guide;
this was followed by the examination of our target in the present study, which is the fleshy
attachment to bone. Accordingly, the present light microscopic results will be represented in
the following order:
B) Enthesis of Tendocalcaneus:
In the present study, sections obtained from the enthesis of tendocalcaneus showed
that it also consisted of the same previously described four zones of biceps brachii. Starting
from the muscle side, zone 1 of dense connective tissue of the tendon, where the collagen
bundles continued with zone 2 of fibrocartilage. Again, zone 2 was followed by zone 3 of
calcified fibrocartilage that was succeeded by zone 4 of compact bone. Further detailed
examination revealed that an irregular dense basophilic line was usually identified between
zone 2 and zone 3 (Fig. 7). Such demarcating line was recognised as a red line in the sections
stained with Masson's Trichrome stain (Fig. 8).
At higher magnification, zone 1 was identified as a region of dense connective tissue
formed of fibroblasts tightly packed between regularly arranged coarse parallel collagen
bundles (Fig. 9). The appearance of chondrocytes with characteristic lacunae indicated the start
of zone 2 of fibrocartilage, while the bundles in the two zones were continuous with each other
(Fig. 9). The chondrocytes were nearly rounded in shape and they were seen arranged either
singly or in clusters (Figs. 9, 10). In addition, the detailed examination of zone 2 demonstrated
that rings of intensely basophilic staining (Fig. 11) frequently surrounded the lacunae of
chondrocytes.
On the other hand, zone 3 was characterized by the presence of rows of chondrocytes
lying singly in their lacunae and surrounded by a deeply stained basophilic matrix (Figs.11,
12). Usually the thickness of zone 2 of fibrocartilage and its population of chondrocytes were
17
evidently greater than those of zone 3 of calcified fibrocartilage (Fig. 10). Moreover, it was
observed that the density of chondrocytes in zone 2 varied from just few rows of cells (Fig. 7)
to large population of irregularly distributed chondrocytes (Figs. 13, 14). In certain regions,
zone 2 of fibrocartilage was not followed by zone 3 of calcified fibrocartilage (Fig. 15).
B) Brachioradialis Muscle:
In the present study, careful examination of the histological sections obtained for the
muscle-bone interface of the attachment of brachioradialis to the lateral supracondylar ridge
again demonstrated three histological zones of tissues (Fig. 20). Zone 1 was identified as the
muscle tissue, formed of skeletal muscle fibres and intervening connective tissue
(endomysium). Zone 2 consisted of dense connective tissue formed of coarse collagen bundles
arranged in different directions. The collagen bundles, toward the muscle, were seen to run in
a plane perpendicular to that of the collagen bundles that faced the bone that ran parallel to it
(Fig. 21). Zone 3 represented the compact bone at the attachment site.
It was noticed that the surface of the bone facing the connective tissue zone (zone 2)
appeared irregular. In addition, the bone matrix close to the site of attachment appeared more
basophilic as compared to the underlying bone tissue (Figs. 20, 21).
18
A) Infraspinatus Muscle:
In the present study, the examined sections of muscle-bone interface of the attachment
of the fleshy fibers of infraspinatus to the infraspinous fossa of the scapula revealed that it
could be divided into three zones. Zone 1 represented the skeletal muscle tissue; zone 2 was
recognised as a layer of connective tissue interposed between zone 1 and zone 3 which was
formed of compact bone (Fig. 27).
Zone 2 of connective tissue commonly appeared to be further subdivided into a part
facing the muscle, formed of dense regularly arranged collagen bundles which was more
fibrous than cellular and a part of less dense collagen bundles, facing the bone. The latter, was
more cellular than fibrous and the collagen bundles were seen running in different directions
19
(Figs. 28, 29).The connective tissue comprising zone 2 appeared to be continuous with that
among the muscle fibers i.e. with endomysium (Fig. 30).
On approaching the bone, it was noted that the surface of the bone sent sawtooth-like
projections toward zone 2 (Fig. 31).
B) Brachialis Muscle:
In the present study, sections of muscle-bone interface of the fleshy origin of brachialis
muscle from the front of the humerus, demonstrated the existence of the same above-mentioned
three zones pattern of tissue described for infraspinatus muscle. However, zone 2 of fibrous
tissue was characteristically more dense and its collagen bundles were seen running parallel to
each other (Fig. 32). As the collagen fibers approached the bone surface, they were attached to
its surface at an acute angle (Fig. 32).
In the present study, the histological structure of muscle-bone interface, where the
muscle pull is transferred to bone, was thoroughly examined in a number of muscles. The
classical pattern of tendon-bone attachment was frequently studied and hence will be discussed
first, followed by dealing with the target of the present study; which is the fleshy-bone interface
examples.
Discussion
I- Tendon-Bony Prominences Attachment (Enthesis):
In the specimens of tendon attached to bony prominences (enthesis), exemplified in
the present investigation by those obtained from the enthesis of biceps brachii muscle at its
insertion into the radial tuberosity, and that of tendocalcaneus at its insertion into the back of
calcaneus, four histological zones were commonly encountered. Such zones were designated
as zone 1, zone 2, zone 3, and zone 4. Starting from the muscle side, zone 1 was identified as
the dense connective tissue of the tendon that was followed successively by zone 2 of
fibrocartilage, zone 3 of calcified fibrocartilage, and lastly zone 4 that was the bone tissue at
the interface. Such sequence and description of these four zones at the attachment site of a
tendon had been previously reported by several authors (Schneider, 1956; Knese and
Biermann, 1958; Biermann, 1975; Benjamin et al., 1991; Benjamin and Ralphs, 2001).
In the present study, zone 2 of fibrocartilage was a constant zone in all specimens
examined, however, zone 3 of calcified fibrocartilage was found to vary from one region to
another of the same enthesis; it was thick in some regions of the tendon, while thin or even
absent in other regions. In that respect, Benjamin and Ralphs (1995) and Evanko and vogel
21
(1990) recognized two types of tendon attachments to bone (enthesis): fibrocartilaginous, and
fibrous. The latter authors proposed that the function of fibrocartilage prevents tendon fibers
bending at the hard tissue interface and thus reduced wear and tear.
Moreover, Salmons (1995) reported that the significance of interweaving collagen
fibers in fibrocartilaginous regions of adult tendons could be purely mechanical, preventing the
tendon from splaying apart when it is under compression against a pulley; like the twisted
strands of a rope. On the other hand, the tendon might be fibrocartilaginous in regions where it
passes around bony pulleys or beneath fibrous retinaculae as an adaptation to resist
compression or shear. Tibialis posterior is fibrocartilaginous where it passes around the medial
malleolus (Vogel et al., 1993) and the extensor tendons of the fingers are similarly modified
where they cross the interphalangeal joints (Benjamin and Ralphs, 1995; Ralphs et al., 1995).
Scanning and electron microscopy of the tibial insertion of patellar ligament of the rat
indicated that fibrocartilage does not follow tendon but merely infiltrates it. So that the tendon
does not diverge during articular movement and the tensile stress is redistributed across the
insertion area by increasing the mechanical coupling among adjacent fibers (Frank et al., 1985;
Raspanti, 1996).
Moreover, Benjamin and Ralphs, (1998) reported that the fibrocartilage of tendons
enthesis is a dynamic tissue that disappears when the tendons are rerouted surgically and can
be maintained in vitro when discs of tendons are compressed. In addition, a wide variety of
extracellular matrix molecules has been reported in enthesis fibrocartilage, particularly type II
collagen and aggrecan, which count for its compression-tolerance properties (Benjamin and
Ralphs, 2001).
Out of the present study, certain structural functional correlation could be assumed.
The well-developed constant zone of the non-calcified fibrocartilage (zone 2) in both biceps
brachii and tendocalcaneus might be related to their high degree and frequency of their
movement during daily activities; where the biceps acts as a supinator and flexor of the elbow,
which are frequently associated with handling objects. In addition, tendocalcaneus glides
against calcaneus with each step during walking or running, so the duration and frequency of
the friction with the bone in these two examples is maximal and consequently the fibrocartilage
is highly needed to minimize wear and tear.
In the support with the above, Benjamin et al. (1994) related the large amount of
fibrocartilage of enthesis of biceps brachii to its wider range of movement as compared with
enthesis of triceps tendons that contained less amount of fibrocartilage. Moreover, Frowen and
Benjamin (1995) reported that tendocalcaneus had a well-developed fibrocartilaginous enthesis
21
as compared to extensor digitorum longus and flexor hallucis longus that were mostly fibrous
enthesis. The authors ascribed the differences between the thickness of fibrocartilage in the
different tendons to differences in the extent to which each tendon is free to move near its
enthesis, the most mobile one is tendocalcaneus has the greatest thickness of fibrocartilage.
In the present investigation, the population of chondrocytes in zone 2 of fibrocartilage
varied from just few rows of cells in some regions to numerous aggregations in other regions.
Such difference of chondrocyte population might reflect regional difference in the force
transmitted by the tendon.
In the current work, an irregular acellular dense line seen as highly basophilic (with
haematoxylin and eosin) was usually identified between zone 2 of fibrocartilage and zone 3 of
calcified fibrocartilage. Such demarcating line was stained red with Masson's trichrome
preparations. Tidemark is supposed to be the term that was given to that line by some authors,
but it was poorly described (Rodeo et al., 1993; Staszyk and Gasse, 2001). The tidemark is a
feature not only in enthesis of tendons but also in the articular cartilage (Havelka et al., 1984)
and in ligament insertions (Gao and Messner, 1996). It could be assumed that the serrated
course of this line creates a sort of interdigitations between zone 2 and 3, which adds to the
strength of the tendon.
It is interesting to mention that literally speaking, the tidemark is a sea-water
phenomenon meaning the mark left by the tidal water. Since this term seems to be more literal
than scientific, a term as serrated basophilic line can be proposed as an alternative that
appears to be more clear and more descriptive.
Redler et al. (1975) reported that using scanning electron microscopy demonstrated
that the tidemark of human articular cartilage comprised three bands of compacted fibrils
running in different directions. However, Oettmeier et al. (1989) mentioned that scanning
electron microscopy of the tidemark between non-calcified and calcified articular cartilage,
appeared as an electron-dense impression between hyaline and calcified cartilage, but the
presence of specific architecture and orientation of collagenous fibers could not be shown.
From the clinical point of view, the understanding of the detailed structure of the
interface between bone and tendon is essential in dealing with traumatic avulsions of tendon
insertion and in the selection of tendons for surgical transfer or joint reconstruction (Benjamin
and McGonagle, 2001). Moreover, disorders at enthesis
pathological ossification of the distal tendon, are common and occur in conditions such as
diffuse idiopathic skeletal hyperostosis (DISH) where they affect also the ligaments of
22
vertebral column (Fouad, 1998). They are also commonly seen as sporting injuries such as
tennis elbow and jumper's knee (Benjamin and Ralphs, 2001).
while the external oblique muscle is a major muscle for expulsive acts, forced expiration,
maintaining the position of abdominal viscera and a flexor to the trunk. i.e. it could be a sort of
structural functional adaptation.
Therefore, it could be suggested that there is a definite positive correlation between the
amount and characteristics of fibrous tissue zone of muscle-bone interface and the thickness
and degree of prominence of the linear elevation of bone.
III- The Muscle-Bone Interface of Muscles Attached by Fleshy Fibers Over a Wide Bony
Area:
In the present study, the histological examination of the fleshy attachments of infraspinatus
and brachialis to the infraspinous fossa and the front of the humerus respectively revealed the
presence of the same above mentioned three histological zones. Zone 1 represented the skeletal
muscle tissue; zone 2 was recognised as a layer of connective tissue interposed between zone
1 and zone 3 which was formed of compact bone. Although both muscle specimens exhibited
the same zonation pattern, yet the fibrous tissue of zone 2 of brachialis was evidently more
compact and dense. Such finding might reflect the difference in the range and force of
movement produced by each muscle.
24
To the best of our knowledge, no references have been found up until now dealing with
the relation between fleshy muscle attachment, specially those muscles with wide bony
attachment area, and the periosteum covering the bone. From the previously mentioned
findings of the present study it could be postulated that fleshy muscle attachment to bone
whether linear or over a wide area is a periosteum mediated attachment. Moreover, at such
attachment sites zone 2 of dense connective tissue represents the periosteum, which was
modified in structure so that the dense connective tissue interposed between the skeletal muscle
fibers, and the bone differed in its density and structure to accommodate the pull of the muscle
fibers.
In that respect, it was observed that the muscle-bone interface of strong muscles
exemplified in the present study by the attachments of brachialis, brachioradialis and external
oblique contained more dense collagen fibers, even fibrocartilage whereas in less powerful
muscles as external intercostals and infraspinatus were thinner and less dense.
Moreover, in all instances, such connective tissue medium appeared to be continuous
with that among the muscle fibers and on approaching the bone, the collagen fibers curved to
be attached to its surface at an acute angle bearing a similarity with sharpey's fibers. Again in
several locations the surface of the bone sent sawtooth like projection to which the collagen
fibers where attached aiding in the fixation of the muscle to the bone.
The absence of fibrocartilage in the above pattern of interfaces might be explained by
the difference in the force of muscle over a wide surface area of bone exerting a minimal
amount of traction per unit area.
CONCLUSION
Three patterns of interfaces could be deduced out of the present study, according to the
number and type of the histological zones; (1) the classical pattern of tendon-bone interface
(enthesis) formed of the four zones, (2) the fleshy pattern of the muscle-bone interface
characterized by absence of fibrocartilage, (3) the third pattern is a mixture of the previous two
patterns.
25
Figures
Fig. (1): Photomicrograph of a section of the enthesis of biceps brachii muscle showing
four zones of different tissues. Zone 1(z1): dense connective tissue; zone 2(z2):
fibrocartilage; zone 3(z3): calcified fibrocartilage; zone 4(z4): compact bone. Note the
irregular dense basophilic line (l) between zone 2 and zone 3.
Haematoxylin and eosin. (x 100).
Fig. (2): Photomicrograph of a section of the enthesis of biceps brachii muscle showing
rows of chondrocytes lying singly in their lacunae among the bundles of collagen
fibers of zone 2(z2). Haematoxylin and eosin. (x 400)
26
Fig. (3): Photomicrograph of a section of the enthesis of biceps brachii muscle showing
that chondrocytes reside within their lacunae forming cell nests of up to four
chondrocytes in the well-developed zone of fibrocartilage. Haematoxylin and eosin.
(x 200).
Fig. (4): Higher magnification of the section shown in Fig. (1) enthesis of biceps brachii muscle
showing that zone 3(z3) consisted of chondrocytes and bundles of collagen embedded in a
dense basophilic calcified matrix. The basophilic matrix of zone 3(z3) disappeared abruptly at
its junction with zone 4(z4) of compact bone. Note the corrugated acellular dense basophilic
line between zone 2(z2) and zone 3(z3). Haematoxylin and eosin. (x 200).
27
Fig. (5): Photomicrograph of a section of the enthesis of biceps brachii muscle showing
that the difference in matrix density between zone 2(z2) and zone 3(z3) was not
evident with Masson's trichrome stain. Note the outstanding red line separating the
two zones. Masson's trichrome. (x 100).
Fig. (6): Photomicrograph of a section of the enthesis of biceps brachii muscle showing
that the thickness of zone 3(z3) of calcified fibrocartilage was not regular being thick
in some regions, while thin or even absent in other regions. Note the wavy line
separating z2 and z3. Haematoxylin and eosin. (x 200).
28
29
31
31
32
33
34
35
Fig. (21): Higher magnification of the previous section of the muscle-bone interface of the
attachment of brachioradialis to lateral supracondylar ridge showing that in zone 2(z2), the
outer collagen bundles (facing the muscle) were seen to be cut in a plane perpendicular to
those of the inner collagen bundles (facing the bone). Note the bone surface irregularities and
the dense basophilia of its matrix close to the attachment site. Haematoxylin and eosin. (x 200).
36
37
Fig. (25): Higher magnification of the field shown in the previous figure of the muscle-bone
interface of the attachment of external oblique fleshy fibers to the outer lip of the iliac crest
showing that on approaching the bone surface the fibrocartilaginous zone 2(z2) acquired
dense basophilia variable in thickness forming a prominent zone, zone 3 (z3) similar to the
calcified fibrocartilaginous zone of enthesis. Haematoxylin and eosin. (x 200).
38
39
41
41
SUMMARY
MORPHOLOGICAL STUDY OF THE MUSCLE- BONE INTERFACE IN MAN
The aim of the present study was to investigate the histological structure of the fleshy
muscle-bone interface in selected limb muscles in man, as compared to that of the enthesis, in
an attempt to clarify the way muscle fibers transmit their contractile force to adjacent bone.
The muscle specimens were taken from biceps and tendocalcaneus as examples for the tendonbone attachment (enthesis), from external intercostal, brachioradialis, and external oblique
muscles as examples for the linear fleshy attachment, and from infraspinatus and brachialis as
examples for the fleshy attachment over a wide area.
The muscle-bone interface specimens were collected form six formalin-fixed dissecting
room elderly male cadavers with no gross pathology. The whole muscle-bone interface was
extracted so that each specimen included the muscle and the underlying bone tissues. The
specimens were fixed in 10% neutral buffered formol saline for one week, and then decalcified
with 10% EDTA for about 4-6 weeks. Dehydrated in ascending grades of alcohols, cleared in
xylol, and embedded in paraffin wax. Serial sections were cut at 8-m thickness and stained
with Haematoxylin and eosin, and Masson's trichrome.
In the present work, it was found that tendon-bone attachment of either biceps brachii
or tendocalcaneus was formed of four zones; zone 1 (Z1) of dense connective tissue, zone 2
(Z2) of fibrocartilage, zone 3 (Z3) of calcified fibrocartilage, and zone 4 (Z4) of compact bone.
Serrated basophilic line "tidemark" was usually seen between fibrocartilage and calcified
fibrocartilage zones. Moreover, differences in the distribution and population of chondrocytes
occurred between zone 2 (Z2) and zone 3 (Z3).
On the other hand, the muscle-bone interface of brachialis, infraspinatus,
brachioradialis, and external intercostal muscles was noticed to be formed of three zones; zone
1 (Z1) of skeletal muscle tissue, zone 2 (Z2) of dense connective tissue, and zone 3 (Z3) of
compact bone. The dense connective tissue zone interposed between the skeletal muscle fibers
and the bone differed in its density and structure between the studied muscles. Moreover, some
regions of the attachment site of the external oblique muscle were observed to include zones
of fibrocartilage and calcified fibrocartilage so that a mixture of fibrocartilaginous and fibrous
attachment could be identified.
From the above mentioned findings it was concluded that three patterns of muscle-bone
interfaces could be described according to the number and types of histological zones; (1) the
classical pattern of tendon-bone interface (enthesis) formed of the four zones, (2) the fleshy
pattern of the muscle-bone interface characterized by absence of fibrocartilage, (3) the third
42
pattern is an admixture of the previous two patterns. The present findings would be helpful in
clinical practice; especially, for the choice of the suitable muscle for transplant.
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45
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47