Pathology of Rotator Cuff

Tendonopathy

Bing Wu, MB

Centre for Orthopaedic Research, Department of Surgery

The University of Western Australia

This thesis is presented for the degree of Master of Medical Science

of the University of Western Australia

2008

The work presented in this thesis was performed in the

University of Western Australia Centre for Orthopaedic Research,

Queen Elizabeth II Medical Centre, Nedlands, Western Australia

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Publication and presentation

BING WU, JIMIN CHEN, TAMMYL DELA ROSA, YU QIAN, ALLAN WANG,

JIAKE XU and MING-HAO ZHENG. Autophagic cell death in rotator cuff

tendonopathy. Manuscript in preparation.

JIMIN CHEN, BING WU, TAMMYL DELA ROSA, YU QIAN, ALLAN WANG,

JIAKE XU and MING-HAO ZHENG Autophagic cell death in rotator cuff

tendonopathy: a preliminary result. Proceeding of ASBMR Annual Meeting 2008.

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Contents

Contents ...................................................................................................................................................... 2

Abstract ....................................................................................................................................................... 5

Acknowledgements ..................................................................................................................................... 7

List of figures .............................................................................................................................................. 8

List of tables ................................................................................................................................................ 9

Abbreviations ........................................................................................................................................... 10

1. Introduction .................................................................................................................................... 122

1.1. Anatomy ............................................................................................................................................ 13

1.2. Aetiology ........................................................................................................................................... 15

1.2.1. Extrinsic mechanisms .......................................................................................................... 15

1.2.1.1. Impingement and acromial shape .................................................................................. 15

1.2.1.2. Age ................................................................................................................................. 16

1.2.1.3. Smoking .......................................................................................................................... 17

1.2.2. Intrinsic mechanisms ........................................................................................................... 17

1.2.2.1. Degeneration .................................................................................................................. 17

1.2.2.2. Cuff vascularity .............................................................................................................. 19

1.2.2.3. Neural factors ................................................................................................................ 19

1.2.3. Overuse ................................................................................................................................ 20

1.3. Pathology .......................................................................................................................................... 21

1.3.1. Histology of the normal tendon ........................................................................................... 21

1.3.2. Pathology of tendonopathy .................................................................................................. 23

1.3.3. Healing response .................................................................................................................. 24

1.3.3.1. Acute healing process .................................................................................................... 25

1.3.3.2. Chronic healing process ................................................................................................ 26

1.4. Diagnosis ........................................................................................................................................... 26

1.5. Treatment .......................................................................................................................................... 28

1.5.1. Conservative treatment ........................................................................................................ 28

1.5.1.1. Rest ................................................................................................................................. 28

1.5.1.2. NSAIDs ........................................................................................................................... 28

1.5.1.3. Corticosteroid injections ................................................................................................ 29

1.5.1.4. Exercise .......................................................................................................................... 29

1.5.1.5. Physical modalities ........................................................................................................ 30

1.5.2. Surgical treatment ................................................................................................................ 30

1.5.2.1. Partial-thickness tears ................................................................................................... 31

1.5.2.2. Full-thickness tears ........................................................................................................ 32

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1.6. Summary ........................................................................................................................................... 33

2. Hypothesis and aims ......................................................................................................................... 35

2.1. Hypothesis ......................................................................................................................................... 36

2.2. Aims .................................................................................................................................................. 36

3. Materials and methods ..................................................................................................................... 39

3.1. Materials............................................................................................................................................ 39

3.1.1. Chemical reagents ................................................................................................................ 39

3.1.2. Enzymes............................................................................................................................... 39

3.1.3. Antibodies ............................................................................................................................ 40

3.1.4. Commercial kit .................................................................................................................... 40

3.1.5. Buffer and solutions ............................................................................................................. 40

3.1.6. Other materials .................................................................................................................... 42

3.2. Methods ............................................................................................................................................. 42

3.2.1. Human sample collection .................................................................................................... 42

3.2.2. Tissue preparation ................................................................................................................ 43

3.2.2.1. HE staining .................................................................................................................... 44

3.2.2.2. Immunohistochemistry ................................................................................................... 44

3.2.2.3. TUNEL assay ................................................................................................................. 45

3.2.3. Evaluation of fibril matrix degeneration .............................................................................. 46

4. Results: the variety of cellularity in the disruption of the fibril matrix ....................................... 49

4.1. General description ........................................................................................................................... 50

4.2. Comparisons ...................................................................................................................................... 53

4.2.1. Normal fields vs degenerative fields.................................................................................... 53

4.2.2. Cell density in the different matrix grades ........................................................................... 54

4.3. Discussion ......................................................................................................................................... 54

5. Results: the role of apoptosis in the disruption of the fibril matrix ............................................. 59

5.1. General description ........................................................................................................................... 59

5.2. Comparisons ...................................................................................................................................... 60

5.2.1. Normal fields vs degenerative fields.................................................................................... 63

5.2.2. Percentage of apoptotic cells in the different matrix grades ................................................ 64

5.3. Discussion ......................................................................................................................................... 65

5.3.1. Limitations ........................................................................................................................... 67

6. Results: the role of autophagic cell death in the disruption of the fibril matrix ......................... 70

6.1. General description ........................................................................................................................... 70

6.2. Comparisons ...................................................................................................................................... 73

6.2.1. Normal fields vs degenerative fields.................................................................................... 74

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6.2.2. Percentage of autophagic cell death in the different matrix grades ..................................... 76

6.3. Discussion ......................................................................................................................................... 78

6.3.1. Limitation ............................................................................................................................ 80

7. Results: the role of myofibroblasts in the disruption of the fibril matrix .................................... 82

7.1. General description ........................................................................................................................... 82

7.2. Comparisons ...................................................................................................................................... 84

7.2.1. Normal fields vs degenerative fields.................................................................................... 86

7.2.2. Percentage of myofibroblasts in the different matrix grades ............................................... 87

7.3. Discussion ......................................................................................................................................... 87

8. General discussion ............................................................................................................................ 93

8.1. Future directions................................................................................................................................ 98

9. References ....................................................................................................................................... 101

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Abstract

Tendonopathy, resulting in the loss of mechanical strength of a tendon, is a serious health

problem affecting many people. The common symptom of tendonopathy is pain –

patients‟ daily activities, their participation in sport and exercise, and their ability to work

are greatly compromised. Tendonopathy is considered to be a degenerative disorder

caused by repetitive injury of the tendon. The most common tendon lesions are Achilles

tendon rupture, lateral epicondylitis (tennis elbow) and rotator cuff tear.

However, in spite of its clinical significance, our knowledge about tendonopathy is still

very poor. This research was undertaken to investigate the pathology of tendonopathy. It

is proposed that apoptosis, autophagic cell death and myofibroblasts play a role in the

progression of tendonopathy in the rotator cuff; the aim of this study was therefore to

determine if this was indeed the case.

Tendon tissues were collected from 30 patients suffering from rotator cuff tears. A

terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL assay)

was performed to detect apoptosis. Autophagic cell death of the tenocytes in the ruptured

rotator cuff tendon was detected by immunohistochemical staining for ubiquitin.

Myofibroblasts were identified immunohistochemically with anti-alpha-smooth muscle

actin (anti--SMA) antibody. The distribution of apoptosis, autophagic cell death and

myofibroblasts, as well as the total cell density, were assessed respectively and were

correlated using a four-category (i.e. graded from 0-3) degeneration of collagen matrix.

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The results showed that apoptosis, autophagic cell death and myofibroblasts were

observed in all of the samples. The highest percentage of autophagic cell death was

evidenced in the Grade 2 matrix, while the percentage of apoptosis increased significantly

with the increase of matrix degeneration from Grade 0-3; a similar pattern was found for

myofibroblasts. The total cell numbers varied among the matrix grades, with the

maximum and minimum percentages occurring in Grades 1 and 3, respectively.

It can be concluded that apoptosis, autophagic cell death and myofibroblasts might be

closely related to the damage of the extracellular matrix (ECM) structure.

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Acknowledgements

The work described in this thesis was performed in the Centre for Orthopaedic Research,

School of Surgery, University of Western Australia. I would like to express my sincere

gratitude and appreciation to my two supervisors, Professor Ming-Hao Zheng and

A/Professor Jiake Xu. Without your guidance, support and inspiration, this work would

never have been accomplished. Thank you very much for your constant encouragement.

I would also like to thank Jimin Chen for technical guidance, Yu Qian for statistical

support, and Zhen Lin for useful advice. Many thanks also to my lab colleagues, Dr Sky

Feng, Ms Jamie Tan, Ms Bo Qian, Mr Cheng Loon Leong, Mr Craig Willers, Dr Zi Qiang

Zhou, Mr Ee-Cheng Khor, Ms Estabelle, Dr Felixc Yao, Dr Jacky Chim, Ms Jasreen, Ms

Katherine Mackie, Ms Lesley Gasmier, Ms Pei Ying Ng, Ms Samuel Shee, Mr Tak Sum

and Ms Tamara Davey.

Most importantly, my gratitude to my family. Thank you all so much for the love and

support you have always given me throughout my life. Without you, this thesis would

never have been possible.

Special dedication

This thesis is dedicated to my parents for their unconditional and endless love, guidance

and support throughout my life.

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List of figures

Figure 1.1. The rotator cuff muscles ........................................................................................................ 13

Figure 1.2. Rotator cuff tear types ............................................................................................................ 14

Figure 1.3. The three types of acromions ................................................................................................. 16

Figure 1.4. Possible pathways of tendon degeneration under oxidative stress ......................................... 18

Figure 1.5. The stress versus strain relationship for the progressive loading of a tendon ........................ 20

Figure 1.6. The anatomy of a normal tendon ........................................................................................... 22

Figure 3.1. ECM classification ................................................................................................................. 47

Figure 4.1. ECM grades ........................................................................................................................... 51

Figure 4.2. Blood vessels in torn rotator cuff tendons .............................................................................. 52

Figure 4.3. The torn edge of a tendon ...................................................................................................... 52

Figure 4.4. Comparison of cell density in normal fields and degenerative fields .................................... 54

Figure 4.5. Comparison of cell density in different ECMs....................................................................... 55

Figure 5.1. Apoptotic cells assessed by TUNEL assay ............................................................................ 61

Figure 5.2 Endothelial lining showing apoptosis .................................................................................... 62

Figure 5.3. Comparison of the percentage of apoptotic cells in normal fields and degenerative

fields ...................................................................................................................................... 64

Figure 5.4. Comparison of the percentage of apoptotic cells in different ECMs ..................................... 65

Figure 6.1. A conceptual model of autophagy.......................................................................................... 72

Figure 6.2. Autophagic cell death ............................................................................................................ 74

Figure 6.3. The endothelial lining showing autophagic cell death ........................................................... 75

Figure 6.4. Comparison of the percentage of autophagic cell death in normal fields and

degenerative fields ................................................................................................................. 76

Figure 6.5. Comparison of the percentage of autophagic cell death in different ECMs ........................... 77

Figure 7.1. Myofibroblasts ....................................................................................................................... 84

Figure 7.2. Smooth muscle cells surround blood vessels ......................................................................... 85

Figure 7.3. Comparison of the percentage of myofibroblasts in normal fields and degenerative

fields ...................................................................................................................................... 87

Figure 7.4. Comparison of the percentage of myofibroblasts in different ECMs ..................................... 88

Figure 8.1. A hypothetical model of chronic tendon injury ..................................................................... 97

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List of tables

Table 3.1. Patient data ............................................................................................................................. 43

Table 4.1. Cell density comparison: significance probability (P value) between the four matrix

grades ..................................................................................................................................... 55

Table 5.1. Apoptosis comparison: significance probability (P value) between the four matrix

grades ..................................................................................................................................... 65

Table 6.1. Autophagic cell death comparison: significance probability (P value) between the four

matrix grades .......................................................................................................................... 77

Table 7.1. Myofibroblast comparison: significance probability (P value) between the four matrix

grades ..................................................................................................................................... 88

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Abbreviations

ANOVA Analysis of variance

anti - -SMA anti-alpha-smooth muscle actin

-SMA alpha-smooth muscle actin

DAB Diaminobenzidine

DDW Double distilled water

DEPC Diethylpyrocarbonate

DPX p-xylene-bis-pyridinium bromide

ECM Extracellular matrix

FBS Foetal bovine serum

HE Haematoxylin-eosin

MRI Magnetic resonance imaging

NSAIDs Non-steroidal anti-inflammatory drugs

PBS Phosphate-buffered saline

PCD Programmed cell death

TBS Tris-buffered saline

Tris-HCl Tris (hydroxymehtyl) aminomethane hydrochloride

TUNEL assay Terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling

US Ultrasonography

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Chapter 1

Introduction

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1. Introduction

The rotator cuff is an anatomical term for a group of muscles and their tendons that

stabilise the shoulder by holding the head of the humerus in the small and shallow glenoid

fossa of the scapula. During elevation of the arm, the rotator cuff compresses the

glenohumeral joint, thereby helping the large deltoid muscle elevate the arm further.

Without the rotator cuff, the humeral head would ride up partially out of the glenoid

fossa, and the efficiency of the deltoid muscle would be lessened [105].

A rotator cuff tear is one of the most common injuries seen in orthopaedic practice. The

tendons of the rotator cuff, not the muscles, are most commonly torn. The tear causes

significant pain and restricted movement of the arm, thereby greatly compromising

patients‟ daily activities, their participation in sport and exercise, and their ability to

work. The injury is frequently seen in athletes using repeated overhead motions or

forceful pulling motions such as baseball pitchers, swimmers and tennis players [28, 33,

112, 121, 124, 154, 156, 218].

In the USA, approximately $US7 billion is spent every year on the treatment of shoulder

pain, mainly rotator cuff related injury [163]. In Australia, around 14,000 rotator cuff

repairs are carried out each year, with an estimated cost of $A250 million [Australian

Government Medicare statistics]. More alarmingly, more than 40% of surgery fails

within 12 months, albeit with symptoms relieved [56, 79]. Clearly, this disease affects a

wide range of people, and further studies focusing on this disease and offering potential

for the improvement of treatment options are therefore required.

http://en.wikipedia.org/wiki/Glenoid_fossahttp://en.wikipedia.org/wiki/Glenoid_fossahttp://en.wikipedia.org/wiki/Deltoid_musclehttp://en.wikipedia.org/wiki/Baseballhttp://en.wikipedia.org/wiki/Pitcher

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1.1. Anatomy

The rotator cuff consists of four muscles, subscapularis, supraspinatus, infraspinatus and

teres minor (Figure 1.1). These four muscles are separately attached to the scapula, and

then blend into a single tendon sheet approximately 0.5-0.75 inches from the point of

their attachment to the humerus [57, 68]. The long portion of the bicep adheres to the

glenohumeral capsule and attaches to the humeral head [68].

Figure 1.1. The rotator cuff muscles

The rotator cuff plays an important role in stabilising the glenohumeral joint and rotating

the outside of the humerus [68]. The term „force couples‟ is used to describe the

relationship between intrinsic and extrinsic muscles. The force couples depend on the

function of the rotator cuff [105]. Further, the rotator cuff helps to maintain the integrity

of the joint space and prevents synovial fluid from leaking; it is therefore important in

cartilage nutrition and in the prevention of cuff tear arthropathy [177]. Therefore, any

factor that disrupts the force couples is also likely to contribute to shoulder dysfunction.

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Rotator cuff tears are tears of one or more of the four tendons of the rotator cuff muscles.

Most rotator cuff tears are located in the tendinous part of the cuff, where the tendons

from the corresponding muscles are not individualised [68]. The tears can be classified

into two types, partial-thickness or full-thickness, irrespective of whether they are due to

impingement or to traumatic or degenerative ischaemic aetiology [92]. Patte proposed a

classification system for rotator cuff tears [183] and Ellman‟s classification for their

evaluation is also used in many studies [63, 204] – the quality and mobility of the rotator

cuff tissue is classified by tear pattern, size, retraction and chronicity, and the tear type

(Figure 1.2) is classified by tendon retraction, tear size, cuff mobility and tissue quality

[204].

Figure 1.2. Rotator cuff tear types

A. Transverse tear. B. Anterior L-shaped tear. C. Posterior L-shaped tear. D. Tongue-shaped tear.

E. V-shaped tear. F. U-shaped tear. G. U-shaped tear before and after mobilisation. Figure

courtesy of Sallay et al. [204].

http://en.wikipedia.org/wiki/Tendonhttp://en.wikipedia.org/wiki/Rotator_cuff

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1.2. Aetiology

Despite their frequency and significance, the aetiology of rotator cuff tears is still not

fully understood. There are many factors that contribute to the progression of rotator cuff

disease, these can be grouped into three main areas – extrinsic mechanisms, intrinsic

mechanisms and overuse [178].

1.2.1. Extrinsic mechanisms

Extrinsic mechanism factors include impingement, acromial shape, age and smoking.

1.2.1.1. Impingement and acromial shape

In a study of more than 400 patients with rotator cuff tears, Neer and Poppen state that

95% were caused by impingement associated with lesions, with the anterior third of the

acromion being principally responsible [176, 177]. Normally, the cuff rubs against the

anterior edge of the acromion and the coracoacromial arch when the upper arm is

abducted, flexed and internally rotated. Bigliani et al. [23, 24] related the degree of such

impingements to acromial shape and classified them into three types – type I, flat

acromions, were seen in 17% of cuff tears; type II, curved acromions, in 43% of cuff

tears; and type III, hooked acromions, in 39% of cadaveric full-thickness cuff tears

(Figure 1.3). Further, in a study of more than 200 patients, Wang et al. showed that the

success of conservative management decreased with changes among these three types of

acromion shapes – type I responded in 89% of cases, type II in 73% and type III in 58.3%

(P

– 16 –

Figure 1.3. The three types of acromions

Type I is a flat acromion that provides plenty of room for the rotator cuff; type II is a curved

acromion, leaving less room; type III is hooked acromion, providing little room for rotator

tendon. (Adapted from Jobe CM: Gross anatomy of the shoulder. In Rockwood CA Jr., Matsen

FA III, editors: The Shoulder, Philadelphia, 1990, WB Saunders.)

1.2.1.2. Age

Current studies suggest that acromial shape is age-related. Wang and Shapiro studied the

acromial morphologic characteristics in 272 patients and found a statistical progression

from type I to type III acromion with age [237]. Furthermore, Shah et al. provided

histological and radiographic evidence that acromial shape is acquired through traction

forces rather than congenital abnormalities [210].

Histological analysis has indicated that the incidence and severity of rotator cuff tears

correlate with aging and with the morphology of the acromion [182]. An ultrasound

examination of more than 400 asymptomatic volunteers showed that the frequency

of

rotator cuff tears increased from the youngest group (13%, aged 50-59 years) to the oldest

group (51%, aged 70-79 years) [224]. The same study even suggested that this high tear

prevalence in asymptomatic volunteers should, to a certain extent, be regarded as

– 17 –

„normal‟ degenerative attrition, not necessarily causing pain and functional impairment.

Furthermore, a study of 588 patients found that the average age for patients with no

rotator cuff tear was 48.7 years, rising to 58.7 years for those with a unilateral tear, and to

67.8 years for those with a bilateral tear [253]. All this indicates that age might be one of

the mechanisms for rotator cuff tendonopathy.

1.2.1.3. Smoking

Smoking has also been related to rotator cuff disease, especially in the process of healing.

A 10-year study of 224 patients (95 smokers and 129 non-smokers) who had undergone

rotator cuff repair indicated that the non-smokers had less pain and better results

postoperatively than the smokers [153]. This was also confirmed in a rotator cuff animal

(rat) model, which indicated that nicotine might delay the tendon-to-bone healing process

[80].

1.2.2. Intrinsic mechanisms

Intrinsic mechanism factors include degeneration, cuff vascularity and neural factors.

1.2.2.1. Degeneration

Degeneration is the most common histological finding in tendon ruptures. Investigation

of 891 tendons from acute, subacute and chronic cuff ruptures and 445 control tendons,

showed that 97% of the 891 ruptured tendons presented degenerative changes; the

changes were found in only 33% of the control tendons [118]. Consistent with an earlier

study, the degenerative changes existed in all 74 patients with an Achilles tendon rupture,

suggesting that these changes might result from intrinsic abnormalities before the rupture

[12].

– 18 –

Degenerative changes in tendons might lead to a decrease in tensile strength and a

predisposition to rupture. They are age-related and are typically not seen in younger

individuals [21, 130]. They might also be a result of the accumulation of reactive oxygen

species, thereby leading to oxidative stress and increased apoptotic cells; this has been

implicated in damage to some other tissues [59]. Indeed, an excessive percentage of

apoptosis has been detected in torn rotator cuffs compared with a normal control [230,

256]. This study found evidence that oxidative stress might play an important role in

tendon degeneration by inducing tenocytes to perform apoptosis via a metalloproteinase

within the extracellular matrix (ECM) and c-Jun N-terminal protein kinase within the

intracellular environment [31, 155, 235]. These possible pathways of tendon

degeneration under oxidative stress is outlined in Figure 1.4 [235].

Figure 1.4. Possible pathways of tendon degeneration under oxidative stress

Figure courtesy of Wang et al. [235].

– 19 –

1.2.2.2. Cuff vascularity

In 1934, Codman [48] identified a so-called „critical zone‟ where cuff rupture usually

occurs, located about 1-1.5cm proximal to the humeral insertion of the supraspinatus

tendon [143]. However, histologic and immunohistochemical analyses have reported

relative hyperperfusion at this critical zone [77]. This is supported by intraoperative laser

doppler flowmetry that showed no hypoperfusion in vivo and hyperperfusion at the tear

edge [221]; it was thought that this hyperperfusion resulted from the proliferation in the

subsynovial layer after injury [232]. Based on this, hypovascularity did not appear to be a

reason for rotator cuff tears. However, a possible role for hypovascularity might still

exist. In 1970, Rathbun et al. observed that rotator cuff perfusion reduced markedly when

the arm was fully adducted [190]. In this position, the supraspinatus is compressed at the

humeral head, which might be a contributory factor to hypovascularity. Further, the

assessment of vascular patterns after rotator cuff repair showed that a robust vascular

response after injury decreased with time in the repaired rotator cuff [69]. This study

seemed to show that rotator cuff blood flow had the potential to change in different

conditions, such as before or after injury.

1.2.2.3. Neural factors

A microarray analysis of the supraspinatus tendon in rats after overuse showed increased

expression of a range of glutamate signalling proteins associated with central nervous

system signalling [6, 167]. Further, an increased concentration of substance P was found

in diseased rotator cuffs [89]. It could therefore be hypothesised that neural

overstimulation, in response to overuse, results in the recruitment of inflammatory cells,

– 20 –

in turn leading to painful symptoms and cuff structural disruption, followed by cuff

damage [97]. However, the theory is still not well elucidated.

1.2.3. Overuse

Overuse has been implicated in the aetiology of this disease for a long time and many

studies support this theory. Rees et al. reviewed this as a mechanical theory [192]. The

study proposed that a tendon without any mechanical load has a wavelike structure. Al

loaded tendon, however, passes through two stretch regions. The first is the toe stretch

region; stretching out of its crimped structure, only a small amount of force is required to

straighten it out [53]. Once the force surpasses this range, stretching will continue past the

toe region and enter into the second region, thereby becoming a linear relationship

between strain and force (Figure 1.5) [192]. The strain is directly taken up by the collagen

fibrils and the force versus strain values are therefore determined directly by the

physiological properties of the collagen fibrils.

Figure 1.5. The stress versus strain relationship for the progressive loading of a tendon

This shows three distinct regions (toe, linear and partial failure) prior to complete rupture.

Approximate stress forces (MPa) and strain values (% strain) are shown. Figure courtesy of Rees

et al. [192].

http://rheumatology.oxfordjournals.org/cgi/content/full/45/5/508#FIG3#FIG3

– 21 –

Tendons are composed of two parts, one for low strains and the other for higher strains.

The latter part is usually loaded during locomotion and plays an important role in elastic

energy stores. Strain values of up to 4-8% might be physiological [53, 122, 151, 161, 173,

213]. Within the physiological range, particularly towards a higher range, microscopic

degeneration in the tendon might occur, especially with repeated and/or prolonged

loading. This repeated microtrauma might eventually lead to a decrease in the mechanical

properties of the tendon [20, 53, 170, 252]. Following such microtrauma, the

degenerative tendon is easier to partially rupture, and can lead to a full-thickness tear if

the tendon becomes further weakened [178].

In addition to the above factors, other studies found damage to the tendon can be caused

by overuse and intrinsic injury, overuse and extrinsic compression, as well as overuse

alone [40]. Further, without an additional factor, such as overhead activity, extrinsic

compression alone might be insufficient to cause tendon injury, supporting the role of

multiple factors in the aetiology of some rotator cuff injuries [217]. Overuse might

therefore play an important role in the progression of tendonopathy, with or without

extrinsic and intrinsic factors.

1.3. Pathology

1.3.1. Histology of the normal tendon

A tendon is a tough band of fibrous connective tissue that usually connects muscle to

bone and is capable of withstanding tension. Healthy tendons are brilliant white in colour,

have a fibroelastic texture [211] and are composed of parallel arrays of collagen fibres

http://en.wikipedia.org/wiki/Fibrous_connective_tissuehttp://en.wikipedia.org/wiki/Musclehttp://en.wikipedia.org/wiki/Bonehttp://en.wikipedia.org/wiki/Tension_%28physics%29http://en.wikipedia.org/wiki/Collagen

– 22 –

closely packed together (Figure 1.6). Collagen is arranged in hierarchical levels of

increasing complexity, beginning with tropocollagen, a triple-helix polypeptide chain,

which combines into fibrils, fibres (primary bundles), fascicles (secondary bundles),

tertiary bundles, and the tendon itself [171, 211].

Figure 1.6. The anatomy of a normal tendon

Figure courtesy of Sharma & Maffulli [211].

Most collagen are classified as type I; however, small quantities of other collagen (for

example collagen II, III, IV and V) are also present [47, 215]. The amount of the collagen

fibres is different from tendon to tendon, depending on the location and function of the

tissue; a different mechanical property could lead to a different mixture of these collagen

types [25]. Furthermore, not all collagen arrange in a longitudinal style; collagen fibrils

also transverse each other, providing a good buffer against the longitudinal, transversal,

horizontal as well as rotational forces during movement and capacity [42, 113].

– 23 –

Fibroblast-like cells in tendons known as tenocytes are the only cells that populate

tendons; these are arranged sparsely and evenly in a parallel rows between the parallel

collagen fibrils [194, 203] and respond to cell-matrix interaction [19, 60, 100, 132]. In

haematoxylin-eosin (HE) staining, fibroblasts appear satellite when the tendon is cut as a

cross section, that is they lie between the fibres. In longitudinal sections, fibroblasts are

evenly arranged in parallel rows between the collagen fibrils and the nuclei are typically

flattened and wavy [164].

1.3.2. Pathology of tendonopathy

The term tendinosis was used for many years to describe asymptomatic tendon

degeneration with various histologic features [110, 189]. A number of clinicians still use

the terms tendinitis or tendonitis, implying that the aetiology of this disease is

inflammatory. Nowadays, the term tendonopathy as a generic descriptor of the clinical

conditions in and around tendons is more often used, while the terms tendinosis and

tendinitis are used only after histopathological examination [148].

Tendons that are commonly affected by tendonopathy include the supraspinatus and long

head of biceps in the shoulder, the medial and lateral extensors of the elbow, the patellar,

the Achilles tendon and the posterior tibialis [194]. In most cases, except for the Achilles

tendon, the site affected is at or near the insertion in a fibrocartilaginous region of the

tendon [51]. These sites have common features – they are highly stressed, are often

exposed to repeated strains, including shear or compressive forces, and are relatively less

vascularised [51].

– 24 –

Histological examinations of tendonopathy show loss of fibre organisation, scattered

vascular ingrowth, decreased fibril diameter, changes in cell density (both increased and

decreased), cell rounding, an absence of inflammatory cell, glycosaminoglycan (GAG)

accumulation, hyalinisation, microtears, lipid accumulation and calcification [15, 43,

118, 195, 223]. Collagen I is predominant in the normal tendon, alongside small

quantities of other collagen such as collagen II and III [47, 215]; degenerative tendons are

found to contain much higher levels of collagen III [197]. Indeed, the presence of high

levels of collagen III is a characteristic of the repair process; this will be substituted by

collagen I in normal tendon recovery. Under an electron microscope it can been seen that

collagen fibrils do not form fascicles, do not connect with each other and become

segmented [127]. A repair in tendonopathy is therefore not a total repair; the tendon is

liable to degenerate and it seldom attains the structural integrity and mechanical strength

of normal, healthy tendon [22, 193, 206].

1.3.3. Healing response

Most of the knowledge of tendon healing after injury can be divided into two categories –

acute macrotraumatic injury response and chronic microtraumatic injury response [134].

The former is usually seen in situations of significant tendon trauma as a result of tissue

loss or damage; cumulative effects usually cause the latter. A number of animal studies

have focused on the repair process; however, most are concerned with the acute injury

response, and the chronic response is less understood.

– 25 –

1.3.3.1. Acute healing process

The healing response to acute trauma is generally divided into four overlapping stages. In

the first stage, the injury site is filled with blood clots full of cytokines that recruit

peripheral cells such as polymorphonuclear leukocytes, platelets and lymphocytes into

the wound. These recruited cells expand the inflammatory response and recruit other

types of cells [74, 103].

The next stage starts 24-48 hours later. Macrophages arrive, perform phagocytosis of

necrotic tissues and secrete the growth factors that stimulate neovascularisation and form

granulation tissue. Together with other cells, macrophages release multitype cytokines

such as platelet derived growth factor, epidermal growth factor and basic fibroblasts

growth factor; these trigger the third stage [174, 184].

The third stage of the response starts after 2 days and is characterised by protein

synthesis, including collagen proteins and non-collagen proteins. The tendon, a normally

inactive tissue, becomes a hotbed of cellular activity as the fibroblasts begin to produce

collagen. These fibroblasts are the mixture of the native fibroblasts or tenocytes (the

intrinsic response) and those recruited from the epitenon (the extrinsic response) [201].

Typically, collagen III is deposited followed by the production of collagen I to substitute

collagen III. The total collagen content of the repairing tendon is greater than that of a

normal one, but the concentration of the collagen decreases and the collagen matrix

remains disorganised. This process can last up to 6-8 weeks.

The final stage is associated with the remodelling of the new tendon that matures step by

step. The matrix becomes denser and more oriented longitudinally. The cell population

– 26 –

and synthetic activity decrease and the proportion of collagen I to III begins to approach

normal levels. However, as mentioned previously, the remodelled tissue seldom attains

the structural integrity and tensile strength of a normal, healthy tendon [22, 73, 104, 245].

1.3.3.2. Chronic healing process

The response of tendons to chronic injury differs from that of acute injury. In the chronic

process, degeneration is a significant feature. Normal tendons predominantly contain

collagen I, with a small quantities of collagen III, whilst the degenerative tendon is found

to posses significantly higher levels of collagen III [148, 197]. After the initial injury,

fibroblasts attempt to repair the injury, and collagen III is synthesised. In the acute

healing process, the increased collagen III is replaced by collagen I which is more

resistant to mechanical stress. However, the existing injury factors keep the level of

collagen III production high, which finally results in the failure of shift from the

immature to the mature. This gradually weakens the tendon tissue and eventually

contributes to irreversible tendon injury, causing the chronically injured tendon to rupture

even at a normal physiologic load [94].

1.4. Diagnosis

Tendonopathy is characterised by symptoms such as the gradual onset of morning

stiffness in the injured tendon, dysfunction, localised swelling and sometimes

neovascularisation [30, 121]. Diagnosis is usually based on patient complaints of pain,

alongside palpation of the tendon, its surrounding tissue and its insertion. However,

Fredberg et al. found that diagnosis simply based on clinical examination was, for many

– 27 –

patients, incorrect [75]. Therefore, ultrasonography (US) and magnetic resonance

imaging (MRI), two of the current clinical choices, are recommended for diagnosis,

especially in cases where shooting pain appears or the injury seems to be not responding

to treatment [2, 121].

MRI has been widely investigated for the assessment of shoulder joint diseases as

appropriate surface coils are available [92]. It has been used on athletes‟ posterior glenoid

impingement with a typical, sports-related tendon injury [227]. MRI also has been used to

detect or exclude complete rotator cuff tears with reasonable accuracy, and it is also

suitable to diagnose further pathologies of the shoulder joint [92]. MRI arthrography is

valuable in confirming subtle anatomic details and in further improving the

differentiation of rotator cuff diseases [92].

Compared with MRI, US has several significant advantages. Firstly, the internal

architecture of the examined tendon is more clearly shown than in MRI [116]. Secondly,

US has nearly no limitations in examining the site of injured tendon and any site of

reported pain can be related with real-time scanning, providing a side-to-side comparison

[66, 76]. Thirdly, US can present the neovascularisation associated with this disease. It is

also able to highlight which features of the tendon are as a result of chronic injury. These

include any thickening of the tendon, discontinuity of fibres, focal hypoechoic

intratendinous areas, loss of fascicle organisation, intratendinous focal calcification,

partial or complete ruptures, as well as any thickening of the hypoechoic paratenon with

poorly defined borders, bursitis and adherences between the epitenon and paratenon [27,

71, 86].

– 28 –

1.5. Treatment

Treatment of rotator cuff tears is difficult due to the lack of understanding of the healing

response to chronic trauma. Treatment is therefore based on the assumption that a tendon

responds to chronic injury factors in the same way as it does to an acute injury. The goal

of treatment for rotator cuff disease is to promote healing, eliminate pain and restore

shoulder function. Current treatment options are twofold – conservative and surgical.

1.5.1. Conservative treatment

Conservative treatments are often the priority when the symptom, such as restricted

shoulder function or pain, first appears. Treatments include rest, non-steroidal

anti-inflammatory drugs (NSAIDs), corticosteroid injection, exercise and physical

modalities.

1.5.1.1. Rest

In the early stage, relative rest, therefore avoiding mechanical overload, is an effective

choice to halt the continuing injury, thereby giving the tendon a chance to repair itself.

1.5.1.2. NSAIDs

Anti-inflammatory drugs are used quite commonly to control pain and inflammation.

However, the use of these drugs still remains controversial. In the acute stage, it is

debatable whether blocking the acute inflammatory response is helpful or not, while in

the chronic stage, it is argued that there is little or no inflammatory infiltration [244].

Nevertheless, the drug might have an analgesic effect, possibly independent of the

anti-inflammatory action. Wang & Shapiro found pain was relieved in five out of nine

controlled studies using NSAIDs in the treatment of tendonopathy [237]. The use of

– 29 –

NSAIDs in tendon injuries on animals has also been studied. Some showed the drug

increased tendon tensile strength, while others presented a decrease in mechanical

property [72, 129, 234].

1.5.1.3. Corticosteroid injections

Steroid injections are used in the treatment of tendonopathy for anti-inflammation and

pain relief. Their usage can effectively decrease the pain at the site of injury and can

recover the range of motion exercises to prevent stiffness [26, 55]. However, the

proportion of recurrence is high and, as with NSAIDs, corticosteroid injections remain

controversial. The absence of inflammatory cells, at least in the later stages of

tendonopathy, provides no rational basis to treat the tendonopathy with either NSAIDs or

corticosteroid injection. In addition, this treatment was found to have no effect on the

disease [162] and even to weaken the mechanical property of the tendon [226, 248].

1.5.1.4. Exercise

Exercise therapy has been accepted as one of the mainstays of conservative treatment for

chronic tendon injury and is important in both prevention and treatment of this disease.

Studies show that exercise plays a role in prospective, randomised trials in athletic

patients [5, 150]. Mechanical exercises might stimulate the metabolism of the tenocyte

and promote increased mechanical property, improving the strength and flexibility of the

shoulder [107, 119]; an immobilised tendon could lose up to 40% of it‟s ground substance

[179, 181]. However, it is argued that there is little evidence of a positive effect on

clinical outcomes, such as relief of pain or improvement of function [251].

– 30 –

1.5.1.5. Physical modalities

Physical modalities such as therapeutic ultrasound, laser and heat have been frequently

performed to stimulate collagen production, improve the mechanical properties of the

healing tendon, and alter the local vascular supply to relieve pain [65, 93, 108, 180, 191].

Evaluation of the success of these conservative treatments shows it depends on the

different treatment protocols and the different criteria for the selection of patients; the

percentage varies from less than 50% to about 80% [44, 88, 107, 168, 205, 222].

However, current reviews of these studies show a decrease of the initial successful

percentage due to recurrence on longer follow-ups. The high levels of failure or

recurrence of conservative treatments indicate that rotator cuff tendonopathy is not

self-limiting and that current conservative treatment needs to be improved and developed.

1.5.2. Surgical treatment

Surgery is often considered the last option in the treatment of tendonopathy [9]. Patients

who exhaust all non-operative options and fail to improve after a period tend to be

subjected to the surgery. This period might last between 6-18 months [62, 177]; however,

in some conditions, earlier surgery is indicated, for example, in an acute full-thickness

tear in a younger patient with loss of function [231].

The aim of surgical treatment is to alleviate or eradicate pain and correct functional

deficit [231]. In the operation process, the rupture in the tendon needs to be repaired to

restore the continuity of the tendon, that is, the surgery aims to provide a more suitable

environment for healing of the injured tendon. Removal of a part of the tendon does not

influence the healing process. For example, the mechanical property increased when the

– 31 –

central third of canine patellar tendons were removed [36, 39] and greater cross-sectional

diameters were found in rabbit Achilles tendons after tenotomy [135].

Surgery to repair torn rotator cuffs date back to 1911 [49]. In 1972, Neer reported his

outcomes after studying anterior acromioplasty combined with the release and repair of

torn tendons [176], and emphasised four findings – release of the cuff in order to mobilise

the tendon-muscle unit and a strong attachment to the greater tuberosity, subacromial

release via the division of the coracoacromial ligament and acromioplasty, painstaking

repair of the deltoid, and appropriate postoperative rehabilitation therapy consisting

chiefly of passive movements [68]. These principles are widely accepted and have

contributed to an improvement in surgical techniques. Early primary anatomic repair and

carefully controlled rehabilitation after surgery are the principle treatments for nearly all

rotator cuff tears [185]. Based on this, many surgical techniques have been developed to

restore the structure and function of the damaged rotator cuff, such as acromioplasty,

tendon to bone trough repair, and extensive tendon mobilisation techniques.

1.5.2.1. Partial-thickness tears

The treatment for symptomatic partial-thickness rotator cuff tears is controversial.

Regardless of a decompression for primary impingement or a stabilisation procedure for

instability, correction of the underlying pathology should be the initial focus [231]. There

are several options, including leaving the partial tear alone or addressing it with either a

debridement or a repair, either open or arthroscopically [10, 62, 160, 241]. At present,

acromioplasty seems efficient in treating partial-thickness tears [82, 202].

– 32 –

1.5.2.2. Full-thickness tears

The choice of surgical management for full-thickness rotator cuff tears that have failed

with conservative treatments is multifactorial. Traditionally, the size of the tear is the

predominant factor, but the position of the tear, chronicity, fatty infiltration, and scarring

also play a role in determining appropriate treatment [231]; patients‟ symptoms are

another significant consideration [231]. Tendon repair is the mainstay of the treatment,

with anterior acromioplasty as a common adjunct. Numerous open and arthroscopic

surgeries have now been performed for the repair of full-thickness rotator cuff tears

which have relied on adequate mobilisation of the cuff edges, particularly the

arthroscopic techniques [64, 83, 98, 99]. If the tear is massive and cannot be repaired,

partial repair, local muscle advancement, or distant muscle transfer can be performed [35,

85, 165, 199].

Since 1987, when the arthroscope was first used for the surgical treatment of rotator cuff

tears [61], arthroscopic techniques have improved surgical management. Compared with

open surgery, arthroscopic techniques enable the surgeons to find the extrinsic

impingement signs with higher probability. Under the arthroscope, the subacromial

„roughness‟ such as bursal thickening and subacromial spurs that need to be addressed

surgically in the initial cuff repair can be seen more regularly than open surgical treatment

[246].

Acromioplasty is performed to enlarge the anterior subacromial space to accommodate

the rotator cuff and repair tissue as well as relieve the pain, even if the rotator cuff repair

fails to heal. It has been reported that arthroscopic acromioplasty results in adequate

– 33 –

decompression comparable to the open surgery manner [82, 140, 200]; anterior

acromioplasty, however, does not alter the functional outcome nor the rate of clinic

recurrence after repair [133]. Goldberg et al. reported that 27 full-thickness cuff repairs

without acromioplasty avoided the possibility of deltoid detachment, and altered deltoid

mechanics, anterior-superior instability and tendon scaring to the cancellous undersurface

of the acromion [87]. They concluded that statistically significant improvements in

shoulder comfort and specific shoulder functions could be seen in patients with

full-thickness rotator cuff tears after repair without acromioplasty.

1.6. Summary

Rotator cuff tendonopathy, resulting in the loss of mechanical strength of a tendon, is a

serious health problem affecting many people. In spite of its clinical significance, our

knowledge about tendonopathy is still very poor. Current theories are not concise enough

to explain why the tendon suffers degeneration, nor the histological characteristic of this

disease. Furthermore, neither of the two current treatment options, conservative or

surgical, can repair the tendon to the same structural integrity and tensile strength of a

normal tendon. There is therefore a need for a better understanding of this disease in order

to improve treatment options.

– 34 –

Chapter 2

Hypothesis and Aims

– 35 –

2. Hypothesis and aims

There is a high recurrence rate of tendonopathy due to tendon degeneration. Typical

tendonopathy changes include rounding of nuclei, adipose tissue deposit, vascular

hyperplasia, variation in cellular population and, most importantly, disruption of the

ECM [15, 43, 118, 195, 223]. Tendon ECM is composed largely of type I collagen; this

provides structural integrity and accounts for mechanical strength. The disruption of

collagen fibres, characterised by the loss of longitudinal alignment and the fragmentation

of collagen bundles, directly decreases the mechanical property of tendons and might

eventually result in tendon rupture.

Tendon cell death (apoptosis) has been linked to degeneration of tendon tissue. Recent

studies suggest that apoptosis plays an important role in the development of tendonopathy

[6, 10, 12, 13]. Excessive apoptosis was found in degenerative human tendon tissue and

was considered to be responsible for cell loss and decreased collagen synthesis, resulting

in the progression of tendon degeneration [10, 12]. In addition, another type of

programmed cell death – autophagic cell death – is found to play an important role in

degenerative diseases such as neurodegeneration, muscular disorder and liver conditions

[4, 5, 8, 11]. Therefore, autophagic cell death may also exist and be responsible for the

tendon degeneration to a certain degree. These two types of cell death possibly lead to the

variation of cell number in the ECM, which may be associated with the disruption of the

ECM. Furthermore, in torn tendons, tendon cells were found to differentiate into

myofibroblasts, that is the cells with a contractile ability; this was not the case in normal

– 36 –

tendons [3, 9]. All of these cellular events suggest that the fate of tendon cells might be

related to the process of tendon degeneration.

2.1. Hypothesis

This study was based on three hypotheses, namely that:

Autophagic tendon cell death occurs in a torn human tendon.

Two types of cell death in the injured tendon, autophagic cell death and apoptosis,

play a role in the progression of tendonopathy in rotator cuff.

Cell differentiation into myofibroblasts in the injured tendon plays a role in the

progression of tendonopathy in rotator cuff.

2.2. Aims

The aims of this study were therefore to:

Determine if variety of cellularity was related to the disorganisation of the fibril

matrix – semi-quantitative microscopic analysis was used to determine the

relationship between cell density and the fibril structure with different degrees of

degeneration. This is discussed in Chapter 4.

Determine if rotator cuff tendonopathy was related to apoptosis – TUNEL assay

was used to confirm the existence of apoptosis in torn rotator cuff and

semi-quantitative microscopic analysis was used to determine the relationship

between the percentage of apoptosis and the fibril structure with different degrees

of degeneration. This is discussed in Chapter 5.

– 37 –

Determine if rotator cuff tendonopathy was associated with autophagic cell death –

immunohistochemistry was used to confirm the existence of autophagic cell death

in torn rotator cuff and semi-quantitative microscopic analysis was used to

determine the relationship between the percentage of autophagic cell death and the

fibril structure with different degrees of degeneration. This is discussed in Chapter

6.

Determine if cell differentiation was associated with the process of tendon

degeneration – immunohistochemistry was used to confirm the existence of

myofibroblasts in the torn rotator cuff and semi-quantitative microscopic analysis

was used to determine the relationship between the percentage of myofibroblasts

and the fibril structure with different degrees of degeneration. This is discussed in

Chapter 7.

– 38 –

Chapter 3

Materials and

Methods

– 39 –

3. Materials and methods

3.1. Materials

A number of different materials were used in the study. They are outlined below.

3.1.1. Chemical reagents

The chemical reagents used in the study are listed below.

Chloroform [Merck, VIC, Australia].

3% hydrogen peroxide [chemsupply, Gilman, SA, Australia].

DePeX mounting medium [BDH Chemicals Ltd, Poole, England].

Diaminobenzidine (DAB) [k3468, DAKO, Glostrup, Denmark].

Diethylpyrocarbonate (DEPC) [Sigma Chemical Co. St. Louis, Mo, USA].

Eosin [BDH Chemicals Ltd, Poole, England].

Ethanol [BDH Chemicals Ltd, Poole, England].

Foetal bovine serum (FBS) [Commonwealth Serum Laboratories].

Haematoxylin [Sigma Chemical Co. St. Louis, Mo, USA].

Paraffin wax [BDH Chemicals Ltd, Poole, England].

Phosphate-buffered formalin [BDH Chemicals Ltd, Poole, England].

Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) [Sigma Chemical

Co. St. Louis, Mo, USA].

Xylene [BDH Chemicals Ltd, Poole, England].

3.1.2. Enzymes

The enzymes used in the study are shown below.

– 40 –

Proteinase K [BDH Chemicals Ltd, Poole, England].

Trypsin [ICN Pharmaceuticals, Inc. Costa Mesa, USA].

3.1.3. Antibodies

The antibodies used in the study are shown below.

Anti--SMA primary antibody [M0851, DAKO, Glostrup, Denmark].

Anti-ubiquitin antibody [Z0458, DAKO, Glostrup, Denmark].

Second antibody [K0675, DAKO, Glostrup, Denmark].

3.1.4. Commercial kit

The commercial kit used in the study was the In-situ Cell Death Detection-AP Kit

[1684817 Roche Diagnostics GmbH, Mannheim, Germany].

3.1.5. Buffer and solutions

A number of different buffers and solutions were used. These are outlined below.

95% ethanol: 100% ethanol 950ml/L. Solution was made with DEPC-treated water

and stored at -20oC.

70% ethanol: 100% ethanol 700ml/L. Solution was made with DEPC-treated water

and stored at -20oC.

Alcohol/xylene (1/1): Alcohol 1 volume, xylene 1 volume. Solution was mixed by

gentle inversion and stored at room temperature

DEPC water: DEPC 2ml, absolute ethanol 20ml. DEPC was dissolved in ethanol

and mixed with double distilled water (DDW) to make 2L, incubated at room

– 41 –

temperature in a fume hood overnight, then autoclaved and stored at room

temperature.

1% eosin: 20g eosin was dissolved in 2L 95% alcohol and stored at 4oC.

Gills haematoxylin (1.5L): ethylene glycol 375ml, haematoxylin 6.0g, sodium

iodate 0.6g, aluminium sulphate 42.8g, glacial acetic acid 50ml. Dissolved in

1075ml DDW and stored at room temperature.

Permeabilisation solution (TUNEL) 100ml: 10% triton-X 1ml, 0.1% sodium citrate

99ml. Dissolved in reagents and stored at room temperature.

10% phosphate buffered formalin: Formalin 100ml/L. Diluted in 1xPBS and stored

at room temperature.

20% FBS: DMEM-F/12 400ml, FBS 100ml, L-ascorbic acid 0.5ml,

penicillin-streptomycin (2x) 2.5ml. Solution mixed and stored at 4oC.

PBS (1x): 10xPBS 100ml. Solution mixed with DEPC water, autoclaved and stored

at room temperature.

Proteinase K: Proteinase K 1L, 0.1 M Tris 1ml, EDTA (pH=8.0) 0.05ml. Solution

was fresh and was stored on ice before use.

10mM Tris-HCl (100 ml): 2M Tris-HCl 50L. Diluted in 100ml DEPC water,

autoclaved and stored at room temperature.

Scott‟s tap water: Sodium bicarbonate 7.0g. magnesium sulphate 40g, tap water 2L,

crystal thymol (a few crystals). Mixed and stored at 4oC.

– 42 –

Tris-buffered saline (TBS) (10x): Tris 30.25g, NaCl 42.5g, HCl -17ml, Milli Q

water 500ml pH 7.6 in solution (pH=7.6). Mixed and stored at room temperature.

TBS (1x): 10xTBS 100ml, Milli Q water 900ml. Solution mixed and stored at room

temperature.

3.1.6. Other materials

Other materials used in the study are shown below.

Aperio‟s 120-slide ScanScope XT system [Aperio Technologies, Vista, CA, USA].

Hettich Rotofix 32A Benchtop Centrifuge [HD Scientific Supplies Pty Ltd, NSW,

Australia].

NIS-elements image software [Nikon, Japan].

p-xylene-bis-pyridinium bromide (DPX) permount [14208-10-7 Sigma-Aldrich,

Castle Hill, NSW, Australia].

Reichert-Jung 2030 microtome [Meyer Instruments Inc. TX, USA].

Tissue Tek vacuum infiltration processor [Sakaraus Firetek, USA Inc, USA].

3.2. Methods

3.2.1. Human sample collection

Sample collection was approved by the human ethics committee of UWA. Rotator cuff

tendon samples were harvested from 26 male patients and four female patients (age range

33-74 years; average age 57 years) with rotator cuff tears who were undergoing shoulder

surgery. Patient information included age, sex, lateral side and duration of the disease

(Table 3.1). The ruptured rotator cuffs were obtained 2-84 weeks after rupture. The

– 43 –

samples were sourced from the ragged edges of the torn tendons that were trimmed prior

to surgical repair. No additional tissue was obtained. The samples were fixed, embedded

and cut into 5m thick sections for HE staining, immunohistochemical staining for

ubiquitin and alpha-smooth muscle actin (-SMA), and TUNEL assay.

Table 3.1. Patient data

All data were presented as mean ± SEM.

No. patients (F/M) Age (years) Duration of symptoms (mths) Lateral (right / left)

30 (4/26) 57±2 12±2 22/8

3.2.2. Tissue preparation

The samples harvested during surgery were fixed in 10% phosphate-buffered formalin for

at least 24 hours. All the tissue samples were then processed in a Miles Scientific Tissue

Tek vacuum infiltration processor overnight at 37oC. They were then put in 70% ethanol

for 1 hour, 95% ethanol for 1 hour, 100% ethanol twice for 1 hour each, 100% ethanol

twice for 30 minutes, chloroform twice for 2 hours, chloroform once for 1 hour, and

finally molten wax at 60oC twice for 2 hours, followed by being embedded in paraffin.

The tissues were then cut into 5m thick sections by a Reichert-Jung 2030 microtome,

and floated on a water bath at 40oC; they were then picked up onto slides and dried

overnight at 37oC.

The paraffin-embedded sections were dissolved by treatment in three solutions of 100%

xylene (RNase free) for 2 minutes each. They were then rehydrated by successive

immersion in three changes of 100% ethanol, 95% ethanol and 70% ethanol for 1 minute

each. Finally, the slides were placed in DEPC-treated water.

– 44 –

3.2.2.1. HE staining

The sections were then immersed in Gills haematoxylin for 3 minutes and rinsed in tap

water to remove any excessive stain. The slides were then placed in Scott‟s tap water for 2

minutes and rinsed again in running tap water for 3 minutes. They were then immersed in

70% ethanol and 90% ethanol for 3 minutes each and counterstained with 1% eosin for 1

minute. Finally, dehydration was carried out by immersion in the following solutions in

succession for 3 minutes each – 100% ethanol (three solutions) and absolute

alcohol/xylene (1/1). The sections were cleared in xylene (3 minutes, 3 times) and the

slides were mounted in DePeX and left to dry overnight.

3.2.2.2. Immunohistochemistry

Granular cytoplasmic ubiquitin inclusion by immunohistochemistry can be used as a

measure to detect autophagic cell death in human pathologies on tissue sections [123,

125, 157, 166, 216]. After removal of paraffin and rehydration of the sections, slides were

digested in 0.1% trypsin for 20 minutes, and were then washed by Milli Q water.

Endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 minutes. The

sections were then washed with TBS for 5 minutes. Next, 20% FBS was used to block

non-specific sites on the tissues for 30 minutes and the tissue sections were incubated

with anti-ubiquitin antibody (1:200) for 3 hours at room temperature. Negative controls

were prepared by omitting the primary antibody.

TBS was then used to wash the tissues. Then the second antibody was added into the

tissue for 15 minutes, the tissues were washed by TBS (5 minutes, 3 times), then stained

with DAB for 10 minutes, and were then rinsed off with TBS. Cells positive for

– 45 –

immunohistochemistry showed brown. Finally, the tissues were counterstained in

Mayer‟s haematoxylin, dehydrated through graded alcohol to xylene, and mounted with

DPX permount.

Immunohistochemical staining for myofibroblasts was also performed according to the

same protocol with an anti--SMA primary antibody (1:50). Smooth muscle cells

surrounding blood vessels were used as internal positive control.

3.2.2.3. TUNEL assay

TUNEL assay was used to identify apoptotic cells by labelling nuclear DNA fragments.

The procedure of TUNEL assay is described as follows. After removal of paraffin and

rehydration of the sections, sections were incubated with proteinase K (20mg/ml in

10mM Tris/HCl, pH 7.4-8.0 for 15-30 minutes at 21-37oC), then incubated in 0.1% Triton

X-100 in 0.1% sodium citrate for 8 minutes, and rinsed off twice with PBS. The TUNEL

reaction was prepared in accordance with the instructions from the In-situ Cell Death

Detection-AP Kit. 50L TUNEL reaction mixture (enzyme solution: label solution = 1:9)

was added onto each slide; 50L label solution without enzyme solution was added to the

negative control slide. The slides were placed in a humidified chamber for 60 minutes at

37oC.

Next, the slides were rinsed three times in PBS, and 50L Convert-AP (provided in kit)

was added to each slide, followed by another incubation in a humidified chamber for 30

minutes at 37oC. Then they were rinsed again three times in PBS. DAB was added for

colour development, and the slides were incubated for 10 minutes at room temperature.

Cells positive for TUNEL assay showed brown. Finally, they were counterstained in

– 46 –

Mayer‟s haematoxylin, dehydrated through graded alcohol to xylene, and mounted with

DPX permount.

3.2.3. Evaluation of fibril matrix degeneration

All slides were scanned by Aperio‟s 120-slide ScanScope XT system. Aperio‟s digital

pathology information management software and NIS-elements image software, which

were able to zoom freely between 0-40 times magnification, were used to analyse the

digital images. All of the sections were examined by the histopathologists and classified

according to the following criteria [149, 220, 223]:

Grade 0 – normal tendon, straight parallel packed fibres, some with slight waviness.

Grade 1 – slight changes, some separation of fibres, increased waviness, slight loss

of parallel arrangement, discontinuous fibres.

Grade 2 – moderate changes, larger separation of fibres, increased waviness,

moderate loss of parallel arrangement with deterioration of fibres.

Grade 3 – severe changes, total loss of fibre structure and hyalinisation, total loss of

parallel arrangement, no discernible orientation of fibres.

Under x20 magnification, 30 fields (0.183mm2 each) (Figure 3.1) were randomly selected

from each section. It was difficult to classify the ECM in the section detected by

immunohistochemistry or TUNEL assay, therefore the same area in the HE-staining

section from the same sample was used (Figure 3.1).

– 47 –

Figure 3.1. ECM classification

The same areas in the HE-staining section (red) were used to classify the ECM in the section

detected by immunohistochemistry or TUNEL assay. Each green box presented one chosen area

for analysis (0.183mm2 each).

The process of grading the collagen matrix structure was performed by two independent

individuals. The boundary between the grades has some impact on the assessment, but the

final assessment was consistent. The respective numbers of positive cells for apoptosis,

autophagic cell death and myofibroblasts, as well as the total tenocyte number in each

chosen field, were counted. The percentage of positive rate was calculated as: (positive

cells number / total cell number) x 100. In addition, cell density (cells/mm2) was also

reported as: number of the total cells from the chosen field / the area of analysis.

All the values in the test and figures were expressed as mean ± SEM; statistical analysis

among the different graded areas in the samples was performed using analysis of variance

(ANOVA) [SPSS 16.0] [125, 256]. The confidence limit was predetermined at a

probability level of p

– 48 –

Chapter 4

Results: the variety of

cellularity in the

disruption of the fibril

matrix

– 49 –

4. Results: the variety of cellularity in the disruption of the

fibril matrix

Rotator cuff tendonopathy, resulting in the loss of mechanical strength of tendon, is

considered to be a degenerative disorder characterised by the disruption of the fibril

matrix. The aim of this study was to investigate the relationship between this disruption

and the variety of cellularity in the degeneration of rotator cuff. Tendon tissues were

collected from 30 patients suffering from rotator cuff tears and HE staining was used to

analyse the degree of degeneration of their fibril matrix structure. The distribution of cell

density was assessed, and was correlated with the disruption of fibril matrix, which was

graded on a scale of 0-3 using a customised scoring system.

The torn human rotator cuff tendon shows characteristics of degeneration according to

the number of inflammatory cells. The total cell density varied among the fibril matrix,

with maximal cell population (674±27mm-2

) in the Grade 1 section of the matrix, and

with minimal cell population (395±17mm-2

) in the Grade 3 section. A significant

difference existed between all four grades in the matrix. The results indicated that a

variety of cellularity might be closely related to damage of the ECM structure.

Tendons possess a highly organised fibrillar matrix; this is maintained by resident

tenocytes through a continuous process of matrix remodelling [194]. During chronic

injury due to overuse or microtrauma from repetitive loading, the remodelling for repair

is probably mediated by resident tenocytes [212]. However, the damaged tendon heals

very slowly and seldom attains the structural integrity and mechanical strength of a

– 50 –

normal, healthy tendon [22]. The immature collagen that cannot be shifted to the mature

leads to a decrease in mechanical property and an increase in the risk for rupture.

Furthermore, chronic injuries also influence the function of tendon cells, affecting the

rate of collagen synthesis. Eventually the tendon with a chronic injury presents with

disruption of collagen fibre, one of the most important features of tendonopathy.

The initial phase of this research used a semi-quantitative classification to investigate the

histopathology of the fibril matrix structure in order to find out the relationship between a

variety of cellularity and different degrees of degenerative fibril matrix structure.

4.1. General description

The ruptured rotator cuff samples demonstrated in HE staining revealed a highly variable

collagen structure and cellular morphology. Areas where the fibres were highly organised

were also the areas where the most cells exhibited normal spindle shapes and lined in a

parallel pattern (Figure 4.1 A, A1, A2). Conversely, the cells in the areas where the fibril

matrix were disorganised were more likely to present rounded in shape and lined

randomly (Figure 4.1 B-D). The number of rounded and plump nuclei was numerous

compared to the normal spindle-shaped nuclei. In addition, there was no presence of

neutrophils, macrophages or lymphocytes, and there was an almost total absence of

inflammatory cells.

– 51 –

Figure 4.1. ECM grades

A. Grade 0, representing nearly normal. B. Grade 1. C. Grade 2. D. Grade 3, showing the most

abnormal. A1, B1, C1, D1 The boxed areas (black) in Figure A, B, C, D are shown at a higher

magnification. A2, B2, C2, D2 The boxed areas (black) in Figure A1, B1, C1, D1 are shown at a

higher magnification. Bar is 50m in all pictures.

Further, proliferation of small blood vessels was seen in the majority of samples (21 out

of 30), the normal parallel pattern of the blood supply was lost and the vessels seemed

randomly oriented (Figure 4.2). A number of the small vessels were surrounded by

uncharacteristic circular patterns of collagen matrix. The edges of the tears were lined by

a layer of fibroblasts oriented at an angle to the normal orientation of the tendon fibre and

fibroblast-like cells, and paralleled to the edge of the torn tendon (Figure 4.3). These

layers were frequently in hypercellularity and consisted of rounded and / or

spindle-shaped cells.

– 52 –

Figure 4.2. Blood vessels in torn rotator cuff tendons

Blood vessels are arrowed.

Figure 4.3. The torn edge of a tendon

It can be seen where tendon cells have formed a dense layer of cells.

– 53 –

Collagen structure was noted as a lack of normal parallel pattern, and was graded from

0-3 based on a semi-quantitative four-point grading scale (Figure 4.1 A-D). A mixture of

at least two grades was observed from each section, and no slide was homogenous with a

single grade in all chosen areas. In the 900 chosen fields (30 fields from each sample),

there were 57 fields in the Grade 0 section of the matrix, 246 in the Grade 1 section, 358

in Grade 2, and 239 in Grade 3. In the Grade 0 section, nearly all the tenocytes were lined

in parallel orientation with spindle-shaped nuclei. As the grade for the collagen matrix

structure increased, the distribution of tendon cells, mostly with rounded or

plump-shaped nuclei, lost the parallel arrangement throughout the tissue and had no

discernible pattern (Figure 4.1 A-D).

4.2. Comparisons

4.2.1. Normal fields vs degenerative fields

In order to investigate the distribution of tendon cells in tendonopathy, the chosen fields

were divided into two groups. Group I consisted of those fields with a normal

arrangement of fibril matrix, namely the areas that were graded 0 (57 fields). Group II

was composed of the remaining fields (843 fields) that showed cell degeneration, from a

slight change to total deterioration. The mean cell density in areas with absence of

degeneration was 555±41, while the fields with an abnormal fibril structure presented a

comparatively lower result (553±12) (Figure 4.4). It can therefore be determined that no

significant difference existed between these two groups (P=0.658).

– 54 –

Figure 4.4. Comparison of cell density in normal fields and degenerative fields

Mean ± SEM. * p

– 55 –

Figure 4.5. Comparison of cell density in different ECMs

Mean ± SEM. * p

– 56 –

such as vacuolisation [118, 134]; this was suggested to drive the tendon cells to have a

different metabolic activity from normal cells.

Besides cell function, the number of cells in the degenerative tendon was also found to be

significantly different from that in the normal tendon, another feature of tendon

degeneration [15, 43, 118, 195, 223]. From the Grade 0 section with a normal aspect, the

cell density increased to reach a peak in the Grade 1 section, and then dropped in the

Grade 2 section and hit the bottom in the Grade 3 section with the worst degeneration

(Figure 4.4). The increased cell numbers in the Grade 1 section was considered a result of

repair – the injury drove the tissue to proliferation to increase collagen synthesis [212].

However, the chronic injury did not stop damaging the tendon tissue, thereby

deteriorating the fibril matrix structure. Thus, many cells did not survive in such harsh

conditions and cell density began to decrease.

Most tendons derive nutrition from the synovial sheath [211]. Blood vessels coming from

the muscle-tendon junction or tendon-bone insertion also provide some part of nutrition

[146]. However, the blood supply to the tendon is considered poorer than that in muscle

or bone. In a normal tendon, the vessels are not present within the fascicles of tendon. In

this study, many samples (21 in 30) presented proliferation of small blood vessels; their

normal parallel pattern was lost and they seemed randomly oriented (Figure 4.2). An

electron microscope found abnormal structures in these vessels, including thick basal

lamina, several layers of lining cells with contractile elements, lipid vacuoles and rich

endoplasmic reticulum [118]. Therefore, it is argued whether these small vessels

contribute to repair.

– 57 –

In 900 chosen fields, most (358 fields) lost their paralleled arrangement, some (239

fields) even lined in a no discernible pattern, only 57 fields looked normal and 246 fields

presented nearly normal. It was supposed that most of samples were taken from the

patients whose rotator cuff were in moderate or even severe degeneration, and that

therefore the entire spectrum of degeneration was not represented in these samples.

In summary, this histological study enabled us to look inside the cellular response of

tendon cells after injury and lead us to further research to investigate the relationship

between the fibril matrix and tendon cells in tendonopathy.

– 58 –

Chapter 5

Results: the role of

apoptosis in the

disruption of the fibril

matrix

– 59 –

5. Results: the role of apoptosis in the disruption of the

fibril matrix

Rotator cuff tendonopathy is considered a degenerative disorder characterised by the

disruption of fibril matrix. One aim of this study was to investigate the relationship

between the disruption of fibril matrix and apoptosis in the degeneration of rotator cuff.

Tendon tissues were collected from 30 patients suffering from rotator cuff tears. A

TUNEL assay was performed to detect apoptosis. HE staining was used to analyse the

degree of degeneration of fibril matrix structure in torn human tendons. The distribution

of apoptosis was assessed, and was correlated with the disruption of fibril matrix, which

was graded from 0-3 using a customised scoring system. The percentage of apoptosis

significantly increased as the fibril matrix degenerated – the percentage was the lowest

(21.4±2.2%) in the Grade 0 section of the matrix, increased in the Grade 1 section

(26.0±1.4%) and became higher in the Grade 2 section (31.0±1.2