MORPHOLOGICAL STUDY OF THE MUSCLE- BONE INTERFACE IN MAN Master thesis 29 9 2004 by Prof. Dr. Hesham N. Mustafa

MORPHOLOGICAL STUDY OF THE MUSCLEBONE 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

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 tendonâ&#x20AC;&#x201C;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

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 wraparound bony pulleys but are not attached to them.

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.

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.

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 ď &#x2030; collagen in the distal biceps tendon (traction tendons) while type ď ? 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 crosslinks 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.

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 softhard 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.

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

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 Î 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. 11

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 bonetendon 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.

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 cartilagesubchondral 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 13

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) 14

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). 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-Âľm 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). 16

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 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).

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 sawtoothlike 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). 18

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 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 â&#x20AC;&#x153;serrated basophilic lineâ&#x20AC;? 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

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, 23

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).

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.

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)

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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-Âľm 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.

References 1. Amiel, D., Frank, C., Harwood, F., Fronek, J. and Akeson, W. (1984): Tendons and ligaments: a morphological and biochemical comparison. J.Orthop. Resear., 1:257-265. 2. Bain, S.D., Impeduglia, T. M. and Rubin, C. T. (1990): Cement line staining in uncalcified thin sections of cortical bone. Stain Technology, 65:159-163. 3. Benjamin, M., Evans, E.J. and Copp, L. (1986): The histology of tendon attachments to bone in man. J. Anat., 149: 89-100. 4. Benjamin, M., Evans, E.J., Donthineni Rao R., Findlay J.A. and Pemberton, D.J. (1991): Quantitative differences in the histology of the attachment zones of the meniscal horns in the knee joint of the man. J.Anat., 177:127-134. 5. Benjamin, M., Newell, R.L.M., Evans, E.J., Ralphs, J.R. and Pemberton, D.J. (1992): The Structure of the insertions of the tendons of Biceps Brachii, Triceps and Brachialis in elderly dissecting room cadavers. J.Anat., 180:327-332. 6. Benjamin, M., Archer, C.W. and Ralphs, J.R. (1994): Cytoskeleton of cartilage cells. Microsc. Res. Tech., 28:372-377. 7. Benjamin, M., Qin, S. and Ralphs, J.R. (1995): Fibrocartilage associated with human tendons and their pulleys. J.Anat., 187:625-633. 8. Benjamin, M. and Ralphs, J.R. (1995): Functional and developmental anatomy of tendons and ligaments .In Repetitive Motion Disorders of the Upper Extremity. American Academy of Orthopaedic Surgeons. pp. 185-203. 9. Benjamin, M. and Ralphs, J.R. (1998): Fibrocartilage in tendons and ligaments, an adaptation to compressive load. J. Anat., 193: 481-494. 10. Benjamin, M., and Ralphs, J.R. (2001): Enthesis-the bony attachments of tendons and ligaments. J. Anat. Embryol., 106:151-157. 11. Benjamin, M. and McGonagle, D. (2001): The anatomical basis for disease localization in seronegative spondyloarthropathy at enthesis and related sites. J. Anat., 199: 503-526. 12. Benjamin, M., Kumai, T., Milz, S., Boszczyk, B. M., Boszczyk, A. A. and Ralphs, J. R. (2002): The skeletal attachment of tendon-tendon â&#x20AC;&#x153;enthesisâ&#x20AC;?. Comp. Biochem. Physiol. A. Mol. Integr. Physiol., 133: 931-945. 13. Biermann, H. (1975): Die Knochenbildung im Bereich periostaler-diaphysarer Sehnen-und Bandansatze. Zeitschrift furZellforschung undmikroskopische Anatomie 46, 635-671. 14. Brandt, K.D., Mankin, H.J. (1993): Pathogenesis of osteoarthritis. In: Textbook of Rheumatology, vol. 2, Kelley, W.N., Harris, E.D., Ruddy, S. and Sledge, C.B. eds. Philadelphia, Saunders. pp. 13551373. 15. Chen, R., Wang, S., Chen, X. and Xiong, S. (2002): A histological and ultrastructual study of the tidemark in human condylar cartilage. Zhonghua kou qiang Yi Xueza Zhi, 37: 425-427. 16. Clark, J. and Stechschulte, D. J. Jr. (1998): The interface between bone and tendon at an insertion site: a study of the quadriceps tendon insertion. J. Anat., 192: 605-616. 17. Cooper, R. R. and Misol, S. (1970): Tendon and ligament insertion. J. Bone and Joint Surg., 52: 120. 18. Drury, R.A.B and Wallington, E.A. (1980): Carlton's Histological Technique. 5th ed., Oxford University Press, New York, pp210-215. 19. Eijden, T.M.,G.J.van, Kouwenhoven, E., Verburg, J. and Weijs, W.A. (1986): A mathematical model of the patellofemoral joint. J.Biomech., 19:219-229. 20. Evans, E.J., Benjamin, M. and Pemberton, D.J. (1990): Fibrocartilage in the attachment zones of the quadriceps tendon and patellar ligament of man. J. Anat., 171:155-162. 21. Evans, E.J., Benjamin, M. and Pemberton, D.J. (1991): Variations in the amount of calcified tissue at the attachments of the quadriceps tendon and the patellar Ligament in man. J. Anat., 174: 145-151. 22. Evanko S. P. and Vogel K. G. (1990): Ultrastructure and proteoglycan composition in the developing fibrocartilaginous region of bovine tendon. Matrex 10, 420-436.

23. Fawcett, D. W. and Bloom, W. (1994): A textbook of histology, 10th ed.,W.B. Saunders Co., Philadelphia, PA. pp. 56-67. 24. Fouad, F.Y.A. (1998): Regional differences of the spines in diffuse idiopathic skeletal hyperostosis (DISH) as evidences on pathogenesis. Menoufia Med. J.; 10(1), January: 281-300. 25. Francois, R.J., Braun, J. and Khan, M. A. (2001): Enthesis and enthesitis: a histopathologic review and relevance to spondyloarthritides. Curr. Opin. Rheumatol., 13: 255-264. 26. Frank, C., Amiel, D., Woo, S.L.Y.and Akeson, W. (1985): Normal ligament properties and ligament healing. Clini. Orthop. Related Resear., 196: 15-25. 27. Frown, P. and Benjamin, M. (1995): Variations in the quality of uncalcified fibrocartilage at the insertions of the extrinsic calf muscles in the foot. J. Anat., 186:417-421. 28. Gao, J., Öqvist, G. and Messner, K. (1994): The attachments of the rabbit medial meniscus: investigation on morphology using image analysis and immunohistochemistry. J. Anat., 185: 663-667. 29. Gao, J and Messner, K. (1996): Quantitative comparison of soft tissue-bone interface at chondral ligament insertions in the rabbit knee joint. J. Anat., 367-377. 30. Gathercole, L. J. and Keller, A. (1991): Crimp morphology in the Fibre-forming collagens. Matrix, 11: 214-234. 31. Gongadze, L. R. (1987): Basophilic line of the articular cartilage in normal and various pathological states. Arkh. Anat. Gistol. Embriol., 92: 52-57. 32. Ham, A.W. and Cormack D.H. (1987): Ham's Histology, 9th ed., J.B. Lippincott, Philadelphia, pp: 196-198. 33. Havelka, S., Horn, S., Spohrova, D. and Valouch, P. (1984): The Calcified-noncalicfied cartilage interface: the tidemark. Acta Biol. Hung., 35: 271-279. 34. Hems, T. and Tillmann, B. (2000): Tendon enthesis of the human masticatory muscles. Anat. Embryol. , 202: 201-208. 35. Hurov, J. R. (1986): Soft-tissue bone interface: how do attachments of muscles, tendons and ligaments change during growth? A light microscopic study. J. Morphol., 189: 313-325. 36. Kannus, P. (2000): Structure of the tendon connective tissue. Scand. J. Med. Sci. Sports., 10: 312-320. 37. Knese, K.H. & Biermann, H. (1958): Die Knochenbildung an Sehnen und Bandansätzen im Bereich ursprünglich chondraler Apophysen. Zeitschrift für Zellforschyng und mikroskopische Anatomie, 49: 142-187. 38. Koch, S. and Tillmann, B. (1994): Vergleichende Untersuchungen der Struktur von Gleitsehnen im Hinblick auf die Inzidenz von Sehenerupturen . Annals of Anatomy, 176 (Suppl.) 44. 39. Koch, S. and Tillmann, B. (1995): The distal tendon of the biceps brachii. Structure and clinical correlations. Anat. Anz., 177: 467-474. 40. Koob, T.J., Clark, P.E., Hernandez, D., Thurmond, F.A. and Vogel, K.G. (1992): Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage. Archives of Biochem. Biophy., 298: 303- 312. 41. Last, R.J. (1999): Last's Anatomy. Regional and Applied (ed. Sinnatamby, C.S.), 10th ed., Churchill Livingstone, Edinburgh. pp 87-90. 42. Oettmeier, R., Abendroth, K. and Oettmeier, S. (1989): Analysis of the tidemark on human femoral heads. I. Histochemical, ultrastructural and micro analytic characterization of the normal structure of the intercartilaginous junction. Acta Morphol. Hung., 37: 155-168. 43. Oettmeier, R., Abendroth, K. and Oettmeier, S. (1989): Analysis of the tidemark on human femoral heads. II. Tidemark changes in osteoarthrosis a histological and histomorphometric study in nondecalcifed preparations. Acta Morphol. Hung., 37: 169-180. 44. Oguma, H., Murakami, G., Takahashi-lwanaga, H., Aoki, M. and Ishii, S. (2001): Early anchoring collagen fibers at the bone-tendon interface are conducted by woven bone formation: light microscope and scanning electron microscope observation using a canine model. J. orthop., 19: 873-880. 45. Ralphs, J.R., Tyers, R.N.S. and Benjamin, M. (1992): Development of the functionally distinct fibrocartilages at two sites in the quadriceps tendon of the rat: the suprapatella and the attachment of the tendon to the patella. J. Anat. Embryol .,185:181-187. 46. Ralphs, J.R., Benjamin, M., Kneafsey, B. and Lewis, A.R. (1995): Fibrocartilages in the capsule of the proximal interphalangeal joint of human fingers in health and disease. Transactions of the Orthop. Resear. Societ., 20:242- 246.

47. Raspanti, M., Strocchi, R., De Pasquale, V., Martini, D., Montanari, C. and Ruggeri, A. (1996): Structure and ultrastructure of the bone/ligament junction. Itali. J.Anat. Embryol., 101:97-105. 48. Redler, I., Mow, V. C., Zimny, M. L. and Mansell, J. (1975): The ultrastructure and biomechanical significance of the tidemark of articulator cartilage. Clin. Orthop., 112: 357-362. 49. Resnick, D. and Niwayama, G. (1981): The Anatomy of the individual joints. In: Diagnosis of bone and joint Disorders, vol.1, Resnick, D. and Niwayama, G. eds. Philadelphia, London, Saunders. pp: 44174. 50. Robbins, J. R., Vogel, K. G. (1994): Regional expression of m RNA for proteoglycans and collagen in tendon. Europ. J. cell Biolo., 64: 264-270. 51. Rodeo, S.A., Arnoczky, S.P., Torzilli, P.A., Hidaka, C. and Warren, R. F. (1993): Tendon-healing in a bone tunnel: a biomechanical and histological study in the dog. J. bone and joint surg., 1795-1803. 52. Rodeo, S.A. (2001): Studies of Tendon-to-Bone Healing: Exploring ways to improve Graft fixation following Anterior curate ligament Reconstruction. J. Bone and joint surg., pp123-127. 53. Rufai, A., Benjamin, M. and Ralphs, J.R. (1992): Development and ageing of phenotypically distinct fibrocartilages associated with the rat Achilles tendon. J. Anat. and Embryol., 186: 611-618. 54. Rufai, A., Ralphs, J. R. and Benjamin, M. (1996): Ultrastructure of fibrocartilages at the insertion of the rat Achilles tendon. J. Anat., 189: 185-191. 55. Sagarriga, V.C., Kavalkovich, K., Wu, J. and Niyibizi, C. (1996): Biochemical analysis of collagens at the ligament-bone interface reveals the presence of cartilage specific collagens. Arch biochem. Biophys., 328:135-142. 56. Salmons, S. (1995): Muscle. In: Gray's Anatomy, Williams, P.L., Berry, M.M., Dyson, M. and Bannister, L.H., 38th ed., Churchill Livingstone, Edinburgh. pp: 738-764.62. 57. Schneider, H. (1956): Zur Struktur der Schnen ansatzzonen. Zeitschrift der Anatomie und Entwicklungsgeschichte, 119: 431- 456. 58. Schiavone, P. A., Fabbriciani, C., Delcogliano, A. and Franzese, S. (1993): Bone-Ligament in interaction in patellar tendon reconstruction of the ACL. Knee surg. sports traumatol Arthrosc. , 1: 4-8. 59. Staszyk, C. and Gasse, H. (2001): The enthesis of the elbow-joint capsule of the dog humerus. Eur. J. Morphol., 39: 319-323. 60. Suzuki, D., Murakami, G., and Minoura, N. (2002): Histology of the bone-tendon interfaces of limb muscles in lizards. Ann. Anat., 184: 363-377. 61. Suzuki, D., Murakami, G. and Minoura, N. (2003): Crocodilian bone-tendon and bone-ligament interfaces. Ann. Anat., 185: 425-433. 62. Tarsney, F.F. (1972): Rupture and avulsion of Triceps. Clini.Orthop. Related Resear., 83:177-183. 63. Tillmann, B. (1992): Rotatorenmanschettenrupturen. Desinsertion der Supraspinatussehne Naht der Supraspinatussehne Spaltung des ligamentum coracoacromiale. Operative Orthopädie und Traumatologie, 4:181-184. 64. Tischer, T., Milz, S., Maier, M., Schieker, M. and Benjamin, M. (2002): An immunohistochemical study of the rabbit suprapatella, a sesamoid fibrocartilage in the quadriceps tendon containing aggrecan. J. Histochem. Cytochem., 50: 955-960. 65. Vogel, K. G., Ördög, A., Pogany, G. and Olah, J. (1993): Proteoglycans in the compressed region of human tibialis posterior tendon and in ligaments. J. Orthop. Resear., 11:68-77. 66. Weiss, A.PC. Kramer, A.A., Barrach, H. J. and Akelman, E. (1994): Variations in the quantity of the type Π collagen in carpal tunnel syndrome. Transactions of the Orthop. Resear. Societ., 19:385389. 67. Woo, S., Maynard, J., Butler, D., Lyon, R.,Torzilli, P., Akeson, W., Cooper, R. and Oakes, B. (1988) : Ligament ,tendon ,and joint capsule insertions to bone .In: Injury and Repair of the musculoskeletal soft tissues. Woo, S.L.Y. and Buckwaletal, J.A. Eds. Park Ridge, Illinois, American Academy of Orthopaedic Surgeons. pp. 133-166.

‫" دراسة شكلية للوصلة العضلية‪-‬العظمية فى االنسان"‬ ‫رسالة مقدمة من‬

‫الطبيب ‪ /‬هشام نعمان عبد الرحيم‬ ‫للحصول الجزئى على درجة الماجستير‬ ‫فى علم التشريح‬

‫المشرفون‬

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

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

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

‫كلية الطب البشرى‬ ‫جامعة عين شمس‬ ‫‪2004‬‬

‫‪46‬‬

‫‪Arabic Summary‬‬ ‫" دراسة شكلية للوصلة العضلية‪-‬العظمية فى االنسان "‬ ‫تهدف الدراسة الحالية الى فحص التركيب النسيجى للوصلة العضلية‪-‬العظمية فى العضالت التى‬ ‫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 ‫العضالت شكل موضع اتصال العضلة حيث اختيرت كل من العضلة الخارجية بين االضالع والعضلة‬ ‫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 ‫وقع االختيار على وتر العضلة ذات الرأسين العضدية والوتر العرقوبى كمثالين التصال العضلة بوتر مع‬ ‫بروز عظمى‪.‬‬ ‫تم جمع عينات للوصلة العضلية‪-‬العظمية من عدد ستة جثث ذكور أدمية مسنة بال عيوب ظاهرة‬ ‫من غرفة التشريح‪ .‬تم تشريح العضالت واستئصال الوصلة العضلية‪-‬العظمية بحيث تحوى كل عينة العضلة‬ ‫والعظم المتصل بها تم تثبيت العينات فى ‪ %11‬فورمالين محايد ثم تم التخلص من الكلس بواسطة االديتا‬ ‫لمدة ‪ 6-4‬أسابيع‪ .‬ثم تلى ذلك تجفيف تدريجى فى الكحول وغمرها فى البارافين وتمت دراسة العينات‬ ‫بالمجهر الضوئى بعد صبغها بالهيماتوكسلين وااليوسين وكذلك صبغة الماسون ترايكروم‪.‬‬ ‫وقد تبين أن التركيب النسيجى للوصلة بين الوتر والعظم لكل من وتر العضلة ذات الرأسين العضدية‬ ‫والوتر العرقوبى يتكون من أربعة مناطق ‪ :‬المنطقة االولى ( ‪ )Z1‬النسيج الليفى الكثيف و المنطقة الثانية‬ ‫( ‪ (Z2‬النسيج الليفى الغضروفى غير المكلس و المنطقة الثالثة ( ‪ (Z3‬النسيج الليفى الغضروفى المكلس و‬ ‫المنطقة الرابعة ( ‪ ) Z4‬النسيج العظمى المدمج‪ .‬وقد لوحظ وجود خط قاعدى الصبغة مسنن بين النسيج‬ ‫الليفى الغضروفى غير المكلس و النسيج الليفى الغضروفى المكلس‪ .‬كما أظهرت النتائج تباين فى توزيع‬ ‫وكثافة الخاليا الغضروفية بين ‪Z2-Z3‬‬ ‫ومن ناحية أخرى أظهر الفحص المجهرى للوصلة العضلية العظمية لمجموعة العضالت المتصلة‬ ‫ظاهريا بألياف لحمية اما لسطح عريض أو اتصاالت طولية بحافة أو حيد أو عرف أنها تتكون بوجة عام‬ ‫من ثالثة طبقات ‪ :‬الطبقة االولى)‪ (Z1‬النسيج العضلى الهيكلى و الطبقةالثانية ( ‪ (Z2‬النسيج الليفى الكثيف‬ ‫و الطبقة الثالثة (‪ )Z3‬النسيج العظمى المدمج‪.‬‬ ‫هذا وقد نوقشت هذة النتائج وعالقتها بقوة الشد فى العضالت المختلفة وكذلك شكل االتصال بالعظم‪.‬‬ ‫كما تم اقتراح تصنيف نسيجى لثالثة أنماط التصال العضل بالعظم‪ .‬وسوف تساعد نتائج هذة الدراسة فى‬ ‫التطبيقات االكلينيكية وخاصة لجراحى العظام فى اختيار واعادة تثبيت العضلة المناسبة فى االصابات‬ ‫والحوادث‪.‬‬

‫‪47‬‬