1

MORPHOLOGICAL STUDY OF THE MUSCLE-

BONE INTERFACE IN MAN"

THESIS

Submitted for the Partial Fulfillment of the Master Degree

"M.Sc." in Anatomy

Presented by

Hesham Noaman Abdelraheem Mustafa M. B. B. Ch.

Supervised by

Prof. Dr. Mostafa Kamel Ibrahim El-Sayed Professor of Anatomy

Head of Anatomy Department,

Faculty of Medicine

Ain Shams University

Prof. Dr. Fouad Yehia Ahmed Professor of Anatomy

Anatomy Department, Faculty of Medicine

Ain Shams University

Assistant Prof. Dr. Hassan Mostafa Serry Professor of Anatomy Assistant

Anatomy Department, Faculty of Medicine

Ain Shams University

Faculty of Medicine

Ain Shams University

2004

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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.

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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

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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 tendon–bone

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

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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.

I-Macroscopic Features of Muscle-Bone Interface: Knese and Biermann (1958) recognized three forms of muscle attachments: (1)

muscles attached by tendons to cartilaginous apophyses where a cartilaginous outgrowth has

preceded the bony one, e.g. the attachment of iliopsoas to the lesser trochanter; (2) Muscles

with circumscribed diaphyseal tendinous attachment to bony prominences e.g. the deltoid

insertion; (3) Muscles with fleshy attachment to the diaphyseal periosteum, e.g. the fleshy

origin of infraspinatus.

Resnick and Niwayama (1981) reported that large amount of bone existed at the

insertion of brachialis to the coronoid process, while the cortical bone tissues at the insertions

of biceps brachii and triceps muscles were much less in thickness. The authors described such

difference in the amount of bone to the function and development of the coronoid process

which provided an important buttress for the elbow and was already bony at birth. However,

the insertion sites of biceps brachii and triceps remained cartilaginous until they were replaced

by bone in childhood.

Amiel et al. (1984) mentioned that the epiphyseal tendons, which leave smooth

markings on normal bones, did so because separation of tissues after maceration occurred at

the tidemark between the calcified and uncalcified zones of tendon fibrocartilage. Moreover,

as blood vessels did not traverse the tendon fibrocartilage plugs, such areas of smooth tendinous

attachment sites were devoid of vascular foramina.

Hems and Tillmann (2000) mentioned that the compressive forces generated where a

tendon presses against a bony or fibrous pulley might lead to modification in the tendon, the

pulley, or both. In that respect, the periosteum of bony grooves or prominences was frequently

modified to form a fibrocartilaginous or even cartilaginous covering forming a thick white

lining that was clearly visible to the naked eye e.g. the groove on the cuboid for the tendon of

peroneus longus. However, a few periostea were purely fibrous e.g. the groove for extensor

pollicis longus at the radius. On the other hand, the presence of fibrocartilage was more

pronounced in the tendons at the ankle than the wrist, probably because the long axis of the

foot is at right angles to that of the leg.

Benjamin and McGonagle (2001) defined classic enthesis as the bony attachment of a

tendon or ligament while the functional enthesis as regions where tendons or ligaments wrap-

around bony pulleys but are not attached to them.

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II-Microscopic Features of Muscle-Bone Interface: Schneider (1956) described the histology of tendons attached to epiphyses as being

formed of four zones: (1) tendon, (2) uncalcified fibrocartilage, (3) calcified fibrocartilage, (4)

bone. Knese and Biermann (1958) added that, histologically the zone of uncalcified

fibrocartilage appeared to be avascular and consisted of chondrocytes and cartilage matrix

lying between bundles of collagen fibers.

Cooper and Misol (1970) reported that microscopically, bone-tendon interface could

be described as being formed of: (1) Tendon: consisted of parallel collagen fibers with

interspersed elongated cells; (2) Fibrocartilage: consisted of collagen bundles in which the cells

arranged themselves in pairs or rows, became round, lied in lacunae of extracellular matrix

between collagen fibers; (3) Mineralized fibrocartilage: consisted of collagen bundles and

many cells surrounded by mineralized matrix.. The authors added that the uncalcified

fibrocartilage was separated from the mineralized fibrocartilage by a blue line (Tidemark)

perpendicular to tendon fibers; (4) Bone: lamellar bone conforms to the irregular contour of

adjacent mineralized fibrocartilage.

Hurov (1986) examined the attachments of popliteus muscle, semitendinosus tendon,

medial collateral knee ligament, and extensor retinaculum histologically in rabbits, and found

that soft structures were inserted principally into fibrous periosteum or perichondrium. An

extensive collagen fiber framework within the cellular periosteum and perichondrium linked

the fibrous periosteum or perichondrium to subjacent bone or cartilage.

Benjamin et al. (1992) mentioned that differences existed in the thicknesses of

fibrocartilage in the different tendons that related well to differences in the extent to which

each tendon was free to move near its enthesis. The most mobile tendon (Achilles) had the

greatest thickness of fibrocartilage whereas the least mobile tendons (those attached to the

phalanges) had largely fibrous attachments.

Ralphs et al. (1992) noticed that one or more prominent basophilic lines (tidemark)

separated the calcified and non-calcified fibrocartilages where chondrocytes were most

numerous on the muscle side of the tidemark. In thick plugs of fibrocartilage, the collagen

fibers often met the tidemark approximately at right angles, e.g. supraspinatus.

Koch and Tillman (1994) identified the presence of fibrocartilage in the distal tendon

of biceps brachii, in supraspinatus and in peroneus longus. In addition, the proximal tendon of

biceps was fibrocartilaginous as it curved over the head of humerus; where it acts as a long

pulley.

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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,

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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.

III-Chemistry of Muscle-Bone Interface: Koob et al. (1992) noticed that tendons placed under increased compressive loading

upregulate the synthesis of large proteoglycans. Benjamin and Ralphs (1995) stated that the

increased glycosaminoglycan content could protect blood vessels in the endotendon from

compression, although compressed regions of tendons are frequently hypovascular.

Koch and Tillmann (1995) studied the structure and clinical correlations of the distal

tendon of the biceps brachii and described the presence of large chondrocyte-like cells inside

the fibrocartilage while, the extracellular matrix was rich in acidic glycosaminoglycans. The

authors found type collagen in the distal biceps tendon (traction tendons) while type

collagen in the tendon fibrocartilage and type III collagen in the gliding surface of the tendon.

In addition, the authors reported the presence of dermatan-sulfate, keratansulfate and

chondroitin-4- as well as chondroitin-6-sulfate. They concluded that there are significant

differences between the extracellular matrix of traction and gliding tendons, which may be

responsible for the location of tendon rupture.

Kannus (2000) studied the structure of the tendon connective tissue and found that

tendons consist of collagen type I and elastin embedded in a proteoglycans-water matrix. These

elements produced by tenoblasts and tenocytes, which are the elongated fibroblasts and

fibrocytes that lie between the collagen fibers. Soluble tropocollagen molecules form cross-

links to create insoluble collagen molecules, which then aggregate progressively into

microfibrils and then into electronmicroscopically clearly visible units, the collagen fibrils.

Chen et al. (2002) studied the histology, histochemistry, and ultrastructure of the

tidemark in the adult human condylar cartilage. The histochemical study revealed the presence

of alkaline phosphatase and calcium and absence of the proteoglycan in the tidemark region.

Ultrastructurally, the authors observed abundance of the membrane-bound matrix vesicles,

crystals of hydroxyapatite and lipid nodule like substances. Such histochemical and

ultrastructure findings were often observed in the load-bearing areas. Moreover, in such areas

wide horizontal fibrils surrounded the whole tidemark region and were seen to interweave with

the collagen fibers that crossed the tidemark to form a net.

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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.

IV-Structural Functional Correlation of Muscle-Bone Interface: Schneider (1956) postulated that the fibrocartilaginous zones within chondral

insertions might prevent fatigue failure by providing a more gradual transition from soft tissue

to the hard bone. Tarsney (1972) reported that the density of calcified tissue at the insertion of

brachialis might explain why avulsions of this tendon are extremely rare compared to avulsions

of the distal tendons of biceps and triceps which are well recognized though uncommon

injuries. Resnick and Niwayama (1981) mentioned that the reason for the large amount of bone

at the insertion of brachialis might relate to the function and the development of the coronoid

process. The latter provided an important buttress for the elbow and was already bony at birth.

Frank et al. (1985) found that the fibrocartilage has a mechanical role diffusing over

the entire attachment site; this minimizes any local concentration of stress. Eijden et al. (1986)

mentioned that the uncalcified zone of fibrocartilage ensured that the tendon fibers did not

bend, or became compressed at a hard tissue interface offering them some protection from wear

and tear. Bain et al. (1990) found that the greatest amount of total calcified tissue at the

attachment of the quadriceps tendon and patellar ligament was found at the insertion of the

tendon. This was the site, which was subjected to the greatest force. Evans et al. (1990)

suggested that the presence of cartilage matrix reduced the wear and tear on tendons and

ligaments.

Evans et al. (1991) stated that the fibrocartilage has a mechanical role, more

fibrocartilage might be at those enthesis where the tendon bends more at its attachment when

the muscle contracts. This is so in tendons at the elbow and knee. Gathercole and keller (1991)

found that the force transmitted to the bone and the amounts of movement allowed at the soft-

hard tissue interface were among the factors likely to influence the proportion fibrocartilage at

an attachment zone. In that, respect a great quantity of fibrocartilage was identified in the

tendon of biceps due to the greater range of movement of this tendon at biceps attachment.

Biceps supinates the forearm and flexes the elbow; brachialis and triceps act in one plane only,

flexing, and extending the elbow. A similar correlation between range of movement and the

amount of fibrocartilage had been reported in the knee.

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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)

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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.

Raspanti et al. (1996) reported that enthesis fibrocartilage increased the mechanical

coupling among adjacent fibers so that the tendon does not splay during articular movement

and the tensile stress is redistributed across the insertion area.

Benjamin and Ralphs (1998) suggested that there is a good correlation between the

distribution of fibrocartilage within an enthesis and the levels of compressive stress; i.e. where

tendons and ligaments are subjected to compression, they are fibrocartilaginous. This occurred

at two sites: where tendons wrap around bony or fibrous pulleys, and in the region where they

attach to bone. Moreover, interweaving of collagen fibers prevent the tendons from splaying

apart under compression. The extracellular matrix contains aggrecan, which allows tendon to

imbibe water and withstand compression. In addition, the complex interlocking between

calcified fibrocartilage and bone contributes to the mechanical strength of the enthesis, whereas

cartilage-like molecules (e.g. aggrecan and type Π collagen) in the extracellular matrix

contribute to its ability to withstand compression.

Kannus (2000) reported that the basic unit of the tendon is the collagen fiber, which

comprises a bunch of collagen fibrils. A bunch of collagen fibers forms a primary fiber bundle,

and a group of primary fiber bundles forms a secondary fiber bundle. A group of secondary

fiber bundles, in turn, forms a tertiary bundle, and the tertiary bundles make up the tendon. A

fine connective tissue sheath called epitenon surrounds the entire tendon. Within one collagen

fiber, the fibrils are oriented longitudinally, transversely and horizontally. The longitudinal

fibers run parallel and cross each other, forming spirals. The function of the tendon is to

transmit the force created by the muscle to the bone. During movements, the tendons exposed

to longitudinal, transversal, and rotational forces. They must be prepared to withstand direct

contusions and pressures. The three dimensional internal structure of the fibers forms a buffer

medium against forces of various directions, thus preventing damage and disconnection of the

fibers.

Benjamin and McGonagle (2001) suggested that the uncalcified fibrocartilage

dissipates the bending of collagen fibers away from the bone, which ensures that a stretched

tendon or ligament does not narrow too close to the bone. Moreover, the fibrocartilage anchors

the tendon/ligament to the bone and enables it to withstand shear. Enthesis fibrocartilage may

be accompanied by sesamoid and periosteal fibrocartilages that similarly protect the enthesis

from wear and tear and dissipates stress.

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V-Clinical Correlation of Muscle-Bone Interface: Tarsney (1972) reported that the density of calcified tissue at the insertion of brachialis

might explain why avulsions of this tendon are extremely rare. The author added that avulsions

of the distal tendons of biceps and triceps are well recognized though uncommon injuries.

Triceps avulsions frequently included a flake of bone, but avulsions of biceps did not, this may

be related to the division of the biceps tendon into two lamina near the attachment zone, which

might produce a weak point in the tendon.

Brandt and Mankin (1993) reported that fibrocartilage differentiation was much more

pronounced in tendons at the ankle than the wrist. This was related to anatomical factors, which

resulted in mechanical differences. Because the long axis of the foot is at right angles to that

of the leg, tendons at the ankle are permanently bent around the bony malleoli and thus

constantly subject 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. In flexion and

extension, tendons contact retinacula and bone alternately but the loading is less than in the

ankle as body weight is not being supported. All these factors mean that less compressive load

is placed on tendon at the wrist. Fibrocartilaginous regions of tendons that wrap around bony

pulleys are inevitably subject to wear and tear. The damage particularly affects the surface of

the tendons and the fissuring and cell clusters in the epitenon are reminiscent of the fibrillation

that occurs in articular cartilage early in osteoarthritis.

Clark and Stechschulte (1998) studied the interface between bone and tendon of the

quadriceps tendon insertion of rabbit and found that traumatic avulsions of ligament or tendon

insertions rarely occurred at the actual interface with bone, because this attachment was strong

or otherwise protected from injury by the structure of the insertion complex. The authors

mentioned that light microscopy (LM) and scanning electron microscopy (SEM) showed that

tendon fibers in the calcified fibrocartilage where they insert into the patellae, unlike tendon

fibers elsewhere, were not crimped. Moreover, SEM identified no specific cement line.

Oguma et al. (2001) described the process of recovery after avulsion at the bone-

tendon interface in canine model by means of light microscopy and scanning electron

microscopy (SEM). At two weeks, tendons, scar tissue, woven bone and lamellar bone were

present at the insertion site. SEM revealed anchoring of collagen fibril bundles of the scar to

the woven bone i.e. interface between soft tissue and hard tissue. By four weeks, the number

of anchoring fibers had increased and a parallel arrangement of fibers was observed. By six

weeks, the anchoring fibers had developed fully and were distributed densely over the interface.

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Rodeo (2001) studied tendon-to-bone healing using a rabbit model in which a

semitendinosus tendon graft transplanted into femoral and tibial bone tunnels to replace the

anterior cruciate ligament (ACL). Marrow cells as well as other marrow-derived cells from the

surrounding tunnel were observed to initiate the healing process 3-7 days following tendon

transplantation. Fibrous tissue was deposited in the interface in the first 7 days, followed by

proliferation of new bone trabeculae along the edge of the tunnel. Moreover, the author

observed the formation of cartilaginous interface between tendon and bone; however, no

distinct tidemark could be identified.

VI-Tidemark in Ligament Insertions and Articular Cartilage: Benjamin et al. (1986) reported a similarity in structure between the calcified zone of

fibrocartilage at the tendon attachment site and the calcified part of articular hyaline cartilage.

It was noticed that, after maceration of the soft tissues, the calcified fibrocartilage was left

attached to the bone at articular surfaces and at the sites of tendon attachment. Moreover, the

authors noticed that the zones of fibrocartilage in tendons whose attachments were particularly

close to an articular surface (such as rotator cuff), were continuous with the periphery of the

articular cartilage.

Gongadze (1987) studied the epiphyses of long bones in man and animals of various

age using histological, histochemical, and histometrical methods. The author found that the

structural-chemical organization of the basophilic line (tidemark) of the articular cartilage

ensures its barrier role and participation in regulating selective permeability. Reconstruction of

the tidemark in the process of physiological aging and in cases of the articular pathology aimed

to preserve its integrity and in this way, a complete differentiation of the non-calcified and

calcified structures secured. Moreover, deflations in the structural-chemical organization of the

tidemark indicated certain disturbances in the state of the system articular cartilage-

subchondral bone.

Oettmeier et al. (1989) defined the tidemark as a boundary between non-calcified and

calcified articular cartilage. Using scanning electron microscopy, the tidemark was

characterized as an electron-dense impression between hyaline and calcified cartilage,

however, the presence of specific architecture and orientation of collaginous fibers could not

be shown. Moreover, concentrations of calcium, phosphorus and sulphur could not be detected

in the tidemark by means of an X-ray micro analyzer.

Oettmeier et al. (1989) have described the changes of the tidemark in osteoarthritis to

be multiform and could be classified into three degrees of severity. Low-grade tidemark

changes were characterized by reduplications of the tidemark and discontinuities of the

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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.

VII-Comparative Anatomy of Muscle-Bone Interface: Suzuki et al. (2002) studied histologically the bone-tendon interface (enthesis) using

the forelimbs of lizards; and described that fibrocartilage-mediated direct insertion at all

epiphyses, whereas periosteum-mediated indirect insertions, were located at the diaphyses. The

author described the morphology of reptilian bone-tendon interface with; (1) various degrees

of absence of the clear fibrocartilage zonation seen in mammals, including the tidemark, (2)

15

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.

Material and Methods I- Choice of Muscle-Bone Interface Specimens: In the present study, the choice of muscle-bone interface specimens was based on the

form of muscle attachment and the shape of the attachment site to the bone as follows:

A- Tendon Attachment to Bony Prominences (Enthesis): Specimens for this mode of muscle attachment were taken from the attachment of

biceps brachii tendon to the radial tuberosity and from the attachment of tendocalcaneus to the

middle of the posterior surface of calcaneus.

B- Linear Fleshy Attachment: Specimens for this mode of muscle attachment were taken from the external

intercostal muscle at the upper border of the rib, from brachioradialis muscle at its origin from

the lateral supracondylar ridge, and the external oblique muscle at its insertion into the outer

lip of the anterior half of the ventral segment of the iliac crest.

C- Fleshy Attachment over a Wide Bony Area: Specimens for this mode of muscle attachment were taken from brachialis muscle at

its origin from the front of the humerus, and from infraspinatus muscle at its origin from the

infraspinous fossa of the scapula.

The muscle-bone interface specimens were collected form six formalin-fixed

dissecting room elderly male cadavers with no gross pathology.

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).

16

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-µm 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:

I- Tendon-Bony Prominences Attachment (Enthesis): Examination of the enthesis or bony prominences of biceps brachii tendon and

tendocalcaneus as examples of this type of tendinous attachment revealed the following:

A) Enthesis of Biceps Brachii Muscle:

In the present investigation, careful light microscopic examination of the sections

obtained from the enthesis of biceps brachii tendon at its attachment to the radial tuberosity

(insertion) revealed that it comprised four zones of different tissues.

Starting from the muscle side, the first zone (Z1) was found to be composed of dense

white fibrous connective tissue of the tendon. This was gradually changed into a second zone

(Z2) of fibrocartilage. Then a third zone of calcified fibrocartilage (Z3) could be identified.

Finally, a fourth zone (Z4) of compact bone was reached at the site of fusion of the tendon with

the bone. An irregular serrated dense basophilic line was frequently identified between zone 2

of fibrocartilage and zone 3 of calcified fibrocartilage (Fig. 1).

Detailed examination of zone 1 of dense white fibrous connective tissue showed that

it was composed of regularly arranged collagen bundles that ran more or less in the same

direction. Spindle-shaped fibroblasts with elongated flattened dark nuclei where dispersed

among the collagen fibres. Such dense connective tissue was gradually replaced by zone 2 of

fibrocartilage, where the fibroblasts became replaced by large and oval or rounded

chondrocytes. The latter were arranged more or less in rows among the bundles of collagen

fibres (Fig. 2). The chondrocytes were observed to reside within their lacunae singly, or

forming cell nests of up to four chondrocytes. The cell nests were found to be present in certain

sections i.e. not homogenously distributed along the whole thickness of the tendon (Figs. 2, 3).

17

At high magnification, zone 3 was found to consist of chondrocytes and bundles of

collagen embedded in a dense basophilic calcified matrix (Fig. 4). However, in sections stained

with Masson's trichrome, such difference in matrix density between zone 2 and zone 3 was not

evident (Fig. 5). Moreover, it was noticed that the thickness of zone 3 was not regular being

thick in some regions of the tendon, while thin or even absent in other regions i.e. its degree of

development differed across the thickness of the tendon (Fig. 6).

An interesting feature was the presence of an irregular corrugated acellular dense

basophilic line of variable thickness, between zone 2 of fibrocartilage and zone 3 of calcified

fibrocartilage (Fig. 4). In sections stained with Masson's trichrome stain, such line appeared as

an outstanding red line separating the above-mentioned two zones (Fig. 5).

The transition from zone 3 to zone 4 was found to be characterized by the

disappearance of the basophilic matrix of zone 3, but the border between the two zones was

found to be wavy giving a form of interdigitations between the two zones (Figs. 4, 5).

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

18

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).

II- Examples of Linear Muscle-Bone Interface: The examination of the obtained specimens of the muscle-bone interface of muscles

attached by fleshy fibers to long narrow bony prominences; namely, border, ridge or crest

revealed the following:

A) The External Intercostal Muscle:

The examination of sections of the muscle-bone interface of the attachment of the

external intercostal to the upper border of the rib showed that it was composed only of three

zones. Zone 1 was considered to be formed of skeletal muscle tissue because of the absent

tendon by the naked eye, zone 2 comprised dense connective tissue, and zone 3 represented the

compact bone at the site of muscle attachment (Fig. 16).

The collagen bundles in zone 2 were observed to run in different directions, the

bundles away from the bone were parallel to it while those adjacent to the bone fuse with it at

an acute angle (Fig. 17). In several instances, pegs of connective tissue were seen dipping into

the bone (Fig. 18). Accordingly, the surface of bone zone 3 appeared irregular with sawtooth-

like projections toward the connective tissue zone (Fig. 19). It is worthy to mention that the

layer of connective tissue (zone 2) could not be seen by the naked eye, and hence the muscle

was classified to have a direct fleshy attachment to bone.

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).

19

C) The External Oblique Muscle:

In the present investigation, the muscle-bone interface of the attachment of the fleshy

fibres of external oblique muscle to the outer lip of the iliac crest, revealed regional variations.

In some regions, the attachment was noticed to be fibrous, while in other regions, it was

observed to be fibrocartilaginous i.e. it was a combination of the previous patterns of interfaces.

The fibrous attachment consisted of three zones; zone 1 comprised the skeletal muscle fibres,

zone 2 was a zone of dense irregular connective tissue intervening between zone 1 and the last

zone (zone 3) of osseous tissue (Fig. 22).

As regard the regions of fibrocartilaginous attachment, it was noticed that it exhibits

the same pattern and sequence of the four zones previously described for the enthesis (Fig.23).

In that respect, the connective tissue surrounding the muscle fibres extended as zone 1 of dense

irregular connective tissue of bundles of collagenous fibres arranged in various directions (Fig.

24). That zone was followed by zone 2 of fibrocartilage formed of bundles of regularly arranged

collagenous fibres and rows of chondrocytes. On approaching the bone surface, the

fibrocartilaginous zone 2 acquired dense basophilia forming a prominent zone of irregular

thickness similar to the early mentioned zone 3 of calcified zone of fibrocartilage of the enthesis

(Fig. 25). Again, an irregular dense basophilic line was identified between the latter two zones

(Fig. 26). Finally, the fourth zone, zone 4, comprised the cortical bone tissue formed of

haversian systems (Fig. 26).

III- Examples of Broad Muscle-Bone Interface: Histological examination of the fleshy muscle-bone interface at a wide area of bone

surface is represented in the present work by the origin of infraspinatus and brachialis, which

revealed the following features:

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

21

(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

22

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 known as enthesopathy i.e.

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

23

vertebral column (Fouad, 1998). They are also commonly seen as sporting injuries such as

tennis elbow and jumper's knee (Benjamin and Ralphs, 2001).

II- The Muscle-Bone Interface of Fleshy Linear Muscle Attachment: In the present investigation, the concept of describing zones of different tissues as

previously mentioned for the tendon-bone interface was adopted for describing the histological

characteristics of muscle-bone interface of muscles attached by fleshy fibers. It is well known

that linear prominences of bone referred to as line, ridge or crest reflect differences in the

force of muscle pull and stress applied to bone at the muscle attachment site (woo et. al., 1988).

The present study showed a common histological pattern for the interfaces that appear by

naked eye as a fleshy muscle-bone attachment (i.e. no tendon could be seen) .Whether the

muscle was attached to a border, a line or a ridge. Such a common histological picture

comprised three zones: zone 1, considered here as skeletal muscle tissue where it replaced the

tendon at the attachment (by naked eye), zone 2, consisted of dense connective tissue, and zone

3, represented the compact bone at the site of muscle attachment. Up to Our knowledge, the

above-mentioned pattern of the fleshy muscle-bone interface has not been previously reported.

Careful examination of specimens obtained from the external intercostal muscle (muscle

attached to a border), brachioradialis (muscle attached to a ridge) and external oblique (muscle

attached to a crest) revealed some differences only in the structure of zone 2.

In case of external intercostal muscle, the collagen bundles in zone 2 appeared running in

different directions and the innermost fibers were commonly seen to join the bone at an acute

angle. However, in zone 2 of the attachment site of brachioradialis to the lateral supracondylar

ridge, the connective tissue was more dense and the collagen bundles, toward the muscle, were

seen to run in a plane perpendicular to that of the inner collagen bundles that faced the bone

and ran parallel to it.

Exceptionally, it was interesting to detect that the external oblique muscle at its attachment

to the iliac crest, was differentiated as fibrous in some regions and fibrocartilaginous in other

regions. In the former regions zone 2 was formed of thick dense irregular connective tissue

bundles arranged in different directions. In the latter regions of fibrocartilaginous attachment,

both calcified and non-calcified fibrocartilage were included so that the classic pattern of the

four zones similar to those described earlier for tendon-bone interface was identified.

The above differences in the characteristics of zone 2 between the three studied muscles

might be related to the differences in the strength of the muscle pull and the variations in the

obliquity of muscle fibers; the external intercostal merely elevates a succeeding rib, the

brachioradialis initiates pronation and supination and is a supplementary flexor to the elbow,

24

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.

IV) Bone Periosteum in Relation to Enthesis and Fleshy Muscle-Bone Interface: As described in basic histological books, bones are invested by a membrane of

specialized connective tissue with osteogenic potency called periosteum. However, the

periosteum covering is lacking on the ends of long bones that are covered by articular cartilage

as well as where tendons and ligaments insert into the bone (Fawcett, 1994). The periosteum

has an outer fibrous layer and an inner osteogenic layer composed of osteoblasts (Ham and

Cormack, 1987). The latter layer, revert to an inactive bone lining cells indistinguishable from

other connective tissue cells, when neither appositional growth nor resorption is occurring.

However, they retain their osteogenic potential and the periosteum is referred to as resting

periosteum (Ham and Cormack, 1987; Fawcett, 1994).

In addition, coarse bundles of collagen fibers from the outer layer of the periosteum

turn inward penetrating the outer circumferential lamellae and extending between the

interstitial lamellae deeper into the bone; such fibers are called sharpey's fibers (Fawcett 1994).

They arise during the growth of the bone when thick bundles of periosteal collagen fibers

become incarcerated in the bone matrix on the subperiosteal deposition of new lamellae. They

serve to anchor the periosteum firmly to the underlying bone and they are especially numerous

near the sites of attachments of tendons to long bones (Fawcett 1994).

25

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.

26

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)

27

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).

28

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).

29

Fig. (7): Photomicrograph of a section of the enthesis of tendocalcaneus showing that it consisted of four different zones of tissues. Zone 1(z1): dense connective tissue; zone 2(z2): fibrocartilage; zone 3(z3): calcified fibrocartilage; zone 4(z4) compact bone. Note that an irregular dense basophilic line (l) was usually identified between zone 2(z2) and zone 3(z3). Haematoxylin and eosin. (x 100).

Fig. (8): Photomicrograph of a section of the enthesis of tendocalcaneus showing dense red line between zone 2 and zone 3. Masson's trichrome. (x 100).

31

Fig. (9): Photomicrograph of a section of the enthesis of tendocalcaneus showing that zone 1(z1) was formed of fibroblasts tightly packed between regularly arranged coarse parallel collagen bundles. The appearance of rows of chondrocytes with their characteristic lacunae indicated the start of zone 2(z2) of fibrocartilage, while the bundles in the two zones were in continuity. Haematoxylin and eosin. (x 200).

Fig. (10): Photomicrograph of a section of the enthesis of tendocalcaneus showing that the chondrocytes were arranged either single, in rows of several cells or in clusters. Haematoxylin and eosin. (x 200).

31

Fig. (11): Higher magnification of the previous section of the enthesis of tendocalcaneus showing that rings of intensely basophilic staining frequently surrounded the lacunae of chondrocytes. Note the row of chondrocytes in zone 4(z4). Haematoxylin and eosin. (x 200).

Fig. (12): Photomicrograph of a section of the enthesis of tendocalcaneus showing rows of chondrocytes in zone 2(z2) and zone 3(z3). Note the dense basophilic matrix of zone 3. Haematoxylin and eosin. (x 400).

32

Fig. (13): Photomicrograph of a section of the enthesis of tendocalcaneus showing that the thickness of zone 2(z2) and its population of chondrocytes were evidently greater than those of zone 3(z3). Masson's trichrome. (x 100).

Fig. (14): Photomicrograph of a section of the enthesis of tendocalcaneus showing numerous aggregations of chondrocytes in zone 2(z2) in contrast with the narrow basophilic z3. Haematoxylin and eosin. (x 200).

33

Fig. (15): Photomicrograph of a section of the enthesis of tendocalcaneus showing that in this region zone 2(z2) of fibrocartilage was not followed by zone 3 of calcified fibrocartilage. Masson's trichrome. (x 100).

Fig. (16): Photomicrograph of a section of muscle-bone interface of the attachment of external intercostal muscle to the upper border of the rib showing that it was formed of 3 zones; zone 1(z1) of skeletal muscle tissue, zone 2(z2) of dense connective tissue, and zone 3(z3) of compact bone. Masson's trichrome. (x 100).

34

Fig. (17): Photomicrograph of a section of the muscle-bone interface of the attachment of external intercostal muscle to the upper border of the rib showing that the collagen bundles of zone 2(z2) run in different directions, the bundles away from the bone are parallel to it while those adjacent to the bone fuse with it at an acute angle. Haematoxylin and eosin. (x 400).

Fig. (18): Photomicrograph of a section of the muscle-bone interface of the attachment of external intercostal muscle to the upper border of the rib showing pegs of connective tissue dipping into of the bone. Haematoxylin and eosin. (x 200).

35

Fig. (19): Photomicrograph of a section of the muscle-bone interface of the attachment of external intercostal muscle to the upper border of the rib showing that the surface of the bone where it abutted on zone 2(z2) appeared irregular with saw tooth-like projections toward the connective tissue of zone 2(z2). Masson's trichrome. (x 400).

Fig. (20): Photomicrograph of a section of the muscle-bone interface of the attachment of brachioradialis to lateral supracondylar ridge of humerus showing 3 zones of tissues; zone 1(z1) of skeletal muscle tissue, zone 2(z2) of dense connective tissue, and zone 3(z3) of compact bone. Haematoxylin and eosin. (x 100).

36

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).

Fig. (22): Photomicrograph of a section of the muscle-bone interface of the attachment of external oblique fleshy fibers to the outer lip of the iliac crest showing a region of fibrous attachment consisting of three zones; zone 1(z1):skeletal muscle fibers; zone 2(z2): dense irregular connective tissue and zone 3(z3):osseous tissue. Masson's trichrome. (x 40).

37

Fig. (23): Photomicrograph of a section of the muscle-bone interface of the attachment of external oblique fleshy fibers to the outer lip of the iliac crest showing that it was composed of four histological zones. Zone 1(z1): dense connective tissue; zone 2(z2): fibrocartilage; zone 3(z3): calcified fibrocartilage; zone 4(z4) compact bone. Haematoxylin and eosin. (x 100).

Fig. (24): Photomicrograph of a section of the muscle-bone interface of the attachment of external oblique fleshy fibers to the outer lip of the iliac crest showing that the connective tissue surrounding the muscle fibers extended as zone 1(z1) of dense bundles of collagen fibers. This was followed by zone 2(z2) of fibrocartilage. Note the regularly arranged bundles of collagen fibers and the rows (r) of chondrocytes in zone 2(z2). Haematoxylin and eosin. (x 100).

38

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).

Fig. (26): Photomicrograph of a section of the muscle-bone interface of the attachment of external oblique fleshy fibers to the outer lip of the iliac crest showing the irregular dense basophilic line(l) between zone 2(z2) and zone 3(z3). Note zone 4(z4) of cortical bone tissue. Haematoxylin and eosin. (x 200).

39

Fig. (27): Photomicrograph of a section of the muscle-bone interface of the attachment of infraspinatus to the infraspinous fossa showing three zones of tissues; zone 1(z1) of skeletal muscle tissue, zone 2(z2) of dense connective tissue, and zone 3(z3) of compact bone. Haematoxylin and eosin. (x 100).

Fig. (28): Photomicrograph of a section of the muscle-bone interface of the attachment of infraspinatus to the infraspinous fossa showing that zone 2(z2) was subdivided into a part facing the muscle (P1) formed of dense regularly arranged collagen bundles and a part facing the bone (P2) of less dense collagen bundles running in different directions. Haematoxylin and eosin. (x 200).

41

Fig. (29): Photomicrograph of a section of the muscle-bone interface of the attachment of infraspinatus to the infraspinous fossa showing that the part facing the muscle (P1) was more fibrous than cellular, while the part facing the bone (P2) appeared more cellular than fibrous. Haematoxylin and eosin. (x 100).

Fig. (30): Photomicrograph of a section of the muscle-bone interface of the attachment of infraspinatus to the infraspinous fossa showing that the collagen bundles of zone 2(z2) (near muscle) were continuous with that among the muscle fibers. Masson's trichrome. (x 200).

41

Fig. (31): Photomicrograph of a section of the muscle-bone interface of the attachment of infraspinatus to the infraspinous fossa showing the sawtooth like projections of the bone surface. Haematoxylin and eosin. (x 400).

Fig. (32): Photomicrograph of a section of the muscle-bone interface of the attachment of brachialis to the front of the humerus showing the dense zone 2(z2) of the collagen fibers. Note that the fibers were attached to the bone at an acute angle. Haematoxylin and eosin. (x 200).

42

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 tendon-

bone 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-µm 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

43

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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46

العظمية فى االنسان"-" دراسة شكلية للوصلة العضلية

رسالة مقدمة من

الطبيب / هشام نعمان عبد الرحيم للحصول الجزئى على درجة الماجستير

علم التشريح فى

المشرفون

االستاذ الدكتور / مصطفى كامل ابراهيم السيد جامعة عين شمس–أستاذ ورئيس قسم علم التشريح ، كلية الطب

االستاذ الدكتور / فؤاد يحيى أحمد جامعة عين شمس–قسم علم التشريح ، كلية الطب -أستاذ علم التشريح

الدكتور / حسن مصطفى سرى جامعة عين شمس–قسم علم التشريح ، كلية الطب -أستاذ مساعد علم التشريح

كلية الطب البشرى

جامعة عين شمس

2004

47

Arabic Summary العظمية فى االنسان "-" دراسة شكلية للوصلة العضلية

تى ال العظمية فى العضالت-تهدف الدراسة الحالية الى فحص التركيب النسيجى للوصلة العضلية

تتصل بالعظم ظاهريا بألياف لحمية وكذلك للعضالت التى تتصل بالعظم من خالل الوتر وذلك فى محاولة

اليضاح الوسيلة التى تنتقل بها قوة الشد من العضل الى العظم. وقد روعى كذلك عند اختيار عينات

ة الع والعضلالعضالت شكل موضع اتصال العضلة حيث اختيرت كل من العضلة الخارجية بين االض

العضدية الكعبرية وعضلة البطن المنحرفة الخارجية كأمثلة على االتصال اللحمى بحافة أو حيد أو عرف

عظمى بالتتابع كل من العضلة العضدية والعضلة تحت شوكة الكتف كمثالين لالتصال بباحة عريضة. كما

ر مع وبى كمثالين التصال العضلة بوتوقع االختيار على وتر العضلة ذات الرأسين العضدية والوتر العرق

بروز عظمى.

العظمية من عدد ستة جثث ذكور أدمية مسنة بال عيوب ظاهرة -تم جمع عينات للوصلة العضلية

ة العضلة العظمية بحيث تحوى كل عين-من غرفة التشريح. تم تشريح العضالت واستئصال الوصلة العضلية

فورمالين محايد ثم تم التخلص من الكلس بواسطة االديتا %11 والعظم المتصل بها تم تثبيت العينات فى

البارافين وتمت دراسة العينات وغمرها فىأسابيع. ثم تلى ذلك تجفيف تدريجى فى الكحول 6-4لمدة

بالمجهر الضوئى بعد صبغها بالهيماتوكسلين وااليوسين وكذلك صبغة الماسون ترايكروم.

لوصلة بين الوتر والعظم لكل من وتر العضلة ذات الرأسين العضدية وقد تبين أن التركيب النسيجى ل

( النسيج الليفى الكثيف و المنطقة الثانية Z1والوتر العرقوبى يتكون من أربعة مناطق : المنطقة االولى )

( (Z2 ( النسيج الليفى الغضروفى غير المكلس و المنطقة الثالثة(Z3 النسيج الليفى الغضروفى المكلس و

( النسيج العظمى المدمج. وقد لوحظ وجود خط قاعدى الصبغة مسنن بين النسيج Z4المنطقة الرابعة )

الليفى الغضروفى غير المكلس و النسيج الليفى الغضروفى المكلس. كما أظهرت النتائج تباين فى توزيع

Z2-Z3وكثافة الخاليا الغضروفية بين

وصلة العضلية العظمية لمجموعة العضالت المتصلة ومن ناحية أخرى أظهر الفحص المجهرى لل

ظاهريا بألياف لحمية اما لسطح عريض أو اتصاالت طولية بحافة أو حيد أو عرف أنها تتكون بوجة عام

النسيج الليفى الكثيف Z2)النسيج العضلى الهيكلى و الطبقةالثانية ) (Z1) من ثالثة طبقات : الطبقة االولى

( النسيج العظمى المدمج.Z3و الطبقة الثالثة )

هذا وقد نوقشت هذة النتائج وعالقتها بقوة الشد فى العضالت المختلفة وكذلك شكل االتصال بالعظم.

ى وسوف تساعد نتائج هذة الدراسة ف كما تم اقتراح تصنيف نسيجى لثالثة أنماط التصال العضل بالعظم.

تيار واعادة تثبيت العضلة المناسبة فى االصابات التطبيقات االكلينيكية وخاصة لجراحى العظام فى اخ

والحوادث.