Research Collection

Doctoral Thesis

Structure and function in tendonexperimental studies on the ultrastructural determinants oftendon biomechanical function

Author(s): Rigozzi, Samuela

Publication Date: 2011

Permanent Link: https://doi.org/10.3929/ethz-a-006715894

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ETH Library

Diss. ETH No. 19668

Structure and function in tendon:

experimental studies on the

ultrastructural determinants of

tendon biomechanical function

A dissertation submitted to the

ETH Zurich

for the degree of

Doctor of Sciences

presented by

Samuela Rigozzi

Dipl. Ing. Mat. Sc. EPFL

born 13th June, 1979

citizen of Blenio TI

accepted on the recommendation of

Prof. Dr. Jess G. Snedeker, examiner

Prof. Dr. Ralph Muller, co-examiner

2011

Table of Contents

Acknowledgements iii

Summary v

Riassunto ix

1 Introduction 1

2 Background 7

2.1 An analogue for tendon . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.1.1 Structure function . . . . . . . . . . . . . . . . . . . . . . . . 9

2.1.2 The rope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.2 Tendon function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.2.1 Anatomical role . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.2.2 Tensile behavior . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.3 Tendon structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.3.1 Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.3.2 Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.4 Structure-function models . . . . . . . . . . . . . . . . . . . . . . . . 17

2.4.1 Composite material theory . . . . . . . . . . . . . . . . . . . . 17

2.4.2 Shear-lag theory . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.4.3 Open question . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3 Tissue-organ level 29

3.1 Local strain measurement . . . . . . . . . . . . . . . . . . . . . . . . 31

4 Material level 47

4.1 Collagen fibril morphology . . . . . . . . . . . . . . . . . . . . . . . . 49

4.2 Collagen fibrils’ role in tendons behavior . . . . . . . . . . . . . . . . 67

4.3 PGs’ mechanical role in tendon behavior . . . . . . . . . . . . . . . . 85

5 Synthesis 101

Abbreviations 114

i

List of Figures 117

List of Tables 123

Acknowledgements

When I started my PhD I was passionate and curious about research and wanted

to deeply investigate an open question to give my own contribution to science.

Looking back at all the time that has passed since then, it has not been “just”

research but rather a 360◦ experience to carry in the baggage of my life. First

of all I would like to thank my principal advisor Prof. Dr. Jess G. Snedeker who

initiated the project and handed me the baton to complete the present work. Our

different ways of thinking and working added a high-spirited nature to the whole

research. Special thanks to Prof. Dr. Ralph Muller who has been present from the

beginning of my thesis providing scientific advice and reviews, especially concerning

all aspects of image processing.

I would like to thank the coordinators and collaborators of the Genostem Project,

who gave me input and insight into the biological research that was running parallel

to the mechanical aspects of the work.

I would like to thank those at the Institute for Biomechanics, the Orthopedic

Research Laboratory in Balgrist, the Institute for Biomedical Engineering, and

the Institute of Nanotechnology; particularly Prof. Dr. Andreas Stemmer for his

microscope and Dr. Al Franco-Obregon, coordinator of BEST and fellow spiderman

fan.

I thank all those — past and present — from the old-timer Moussonstrasse lab,

the brave-hearted who shared the basement of ETH-Hongg, the international group

of Balgrist Uniklinik, and the Nanogroup on the top floor of the CLA. Special

thanks to Dr. Kathryn Stok, for her never-ending priceless support, Dr. Mar-

tin Stauber who explained to me all the mysteries of R as well as some VMS tricks,

and Friederike Schulte, my workplace neighbor for the teatime breaks spent together.

Particular thanks to Ursula Luthi and Therese Bruggmann at the Center for

Microscopy and Image Analysis, University of Zurich, for their technical assistance

with the transmission electron microscopy.

Thank you to my family back in the sunny Ticino, for making me laugh and always

giving me a pleasant option to escape the rainy Zurich weather.

Acknowledgements

Thank you to all my friends near and far that make my social life so much fun.

Most particularly, to the ticinesi FAC friends for the entertainment in Zurich, the

old classmates from Lausanne and Stockholm, and the ones that I’ve met during

my PhD. I am also very thankful to Lorenz, Meli and the Swing Kids for helping

me discover the amazing world of Lindy Hop and all the members of HPD for

teaching me how to fly.

Finally, I gratefully acknowledge the financial support of the ETH Zurich, which

has enabled me to live contentedly and complete my work.

April 2011

Samuela Rigozzi

iv

Summary

Tendon is a connective tissue that transmits mechanical loads and forces from muscle

to bone and in doing so enables locomotion and enhances joint stability. It is a

living tissue and responds to mechanical forces by changing its metabolism as well

as its structural and mechanical properties. Injuries to tendon, including overuse

(tendinopathy), affect millions of people in occupational and athletic settings. After

injury, tendon can heal itself; however the healed tendon typically does not reach

the biochemical and mechanical properties of the tendon prior to injury. The actual

healing dynamics of tendon has been reported to be more complicated than other

soft tissues and does not recover to the same degree compared to before the injury.

An injured tendon usually shows scartissue in the repaired area which results in

higher stiffness and tensile strength then the surrounding healthy tissue. In some

cases, even people who had a tendon rupture and were treated with controlled im-

mobilization (i.e. a plaster), had a very high incidence of re-rupture compared to

people who were surgically treated. A comprehensive understanding of the patho-

logical and biomechanical processes that underlie tendon function and pathology is

essential to improved clinical diagnosis and treatment of tendon pathology. For this

reason a deeper investigation of the tendon components is needed to elucidate the

structure-function relationships in tendons.

Tendon structure, composed of collagen fibrils embedded within a proteoglycan

(PG) rich extracellular matrix (ECM), is the key to its mechanical behavior. Colla-

gen fibers are the major constituent of tendon and are believed to bear most of the

tensile load. The PGs are thought to mediate the organization of collagen ultra-

structure and facilitate force transmission along the discontinuous collagen fibrils. A

considerable number of scientific investigations have already been performed to de-

fine the influence of ultrastructure on the mechanical properties of tendon. However,

the large body of published experimental evidence yields conflicting conclusions with

regard to the fundamental ultrastructural determinants of tendon behavior. Thus,

the aim of this thesis is to elucidate these contrasting results by taking a “bottom

up” approach, by which the basic intermolecular forces of binding within the ECM

between the collagen fibrils and PGs are considered, in conjunction with the tensile

Summary

mechanical properties of individual fibrils. This may help reconcile the conflicting

and counterintuitive results that have thus far been observed in laboratory experi-

ments, and will ultimately help inform clinical diagnosis and treatment of connective

tissue disorders. Specifically, a new protocol to mechanically test Achilles tendon

was developed and validated in order to quantify the mechanical contribution of the

PG concentration in tensile load. Tendons were chemically digested of their PG

secondary chains — the glycosaminoglycans (GAGs) — and compared with the na-

tive ones. Results showed that along the longitudinal axis the mechanical behavior

was heterogeneous in tensile load, especially at the muscle and the bone insertion,

highlighting the importance of performing local strain analysis with regard to ten-

sile tendon mechanics. The contribution of PGs to tensile tendon mechanics at

the macroscale was not straightforward and points to a heterogeneous and complex

structure-function relationship in tendon.

Additionally, two inbred strains of mice showing similar macroscopic mechanical

behavior but different elastic modulus were further investigated in their collagen

fibril morphology to understand their mechanical contribution. New image-analysis

tools were designed to parameterize the collagen fibril morphology. The group with

higher elastic modulus, structurally exhibited a larger mean collagen fibril radius,

smaller specific fibril surface (i.e. the perimeter of the fibril in contact with the

non-collagenous ECM), and a lower concentration of GAGs. As in previous studies,

larger collagen fibril radius appeared to be associated with a stiffer tendon, but this

functional difference could also be attributed to reduced potential surface area ex-

change between fibrils and the surrounding PG rich matrix, in which the hydrophilic

GAG side chains may promote inter-fibril sliding.

Finally, the interactions between the PGs and the collagen fibrils were studied

at the ultrascale in their physiological matrix. A new experimental approach com-

bining macroscopic mechanical loading of tendon with a morphometric ultrascale

assessment of longitudinal and cross-sectional collagen fibril deformations was de-

veloped and validated. Tendons with different PG concentration were submitted to

different target strains at the macroscale and chemically fixed. Collagen fibrils were

characterized with atomic force microscopy (AFM). The mechanical contribution of

PG at the ultrascale was quantified by comparing the difference in the collagen fibril

elongation and diameter between native and GAG-depleted tendon. Tendons with

a lower concentration of GAG showed greater elongation in the collagen fibril.

In conclusion, in conjunction with all the specific studies performed in this thesis,

GAG concentration did not significantly influence the overall tendon mechanical

response. However, at the ultrascale GAGs were responsible for changes in collagen

vi

Summary

fibril elongation, which increased at lower GAG concentration. Thus, GAGs seem

not to link the collagen fibrils in order to mechanically transfer forces between them

in tension, but rather hydrate the ECM, and promote collagen fibril sliding under

tension, most likely to prevent damage.

vii

Riassunto

Il tendine e un tessuto connettivo che trasmette i carichi meccanici e le forze dai

muscoli alle ossa e, in tal modo, consente la locomozione e migliora la stabilita

articolare. Si tratta di un tessuto vivente che risponde alle sollecitazioni meccaniche,

modificando il proprio metabolismo e le sue proprieta strutturali e meccaniche. La

lesione del tendine, compreso un uso eccessivo (tendinopatia), colpisce milioni di

persone in un contesto professionale ed atletico. Dopo una lesione, il tendine puo

ristabilirsi spontaneamente, ma generalmente la sua guarigione non raggiunge le

proprieta biochimiche e meccaniche precedenti la lesione. La dinamica effettiva

della guarigione del tendine e nota per essere piu complicata di quella di altri tessuti

molli e per non recuperare la rigidezza sufficiente, come prima della lesione.

Un tendine infortunato mostra, di solito, tessuti cicatrizzati nella zona riparata

che si traducono in una maggiore rigidezza e resistenza alla trazione. In alcuni casi

si e notato che le persone che hanno subito una rottura del tendine e sono state

trattate con immobilizzazione controllata (gesso) hanno avuto un’incidenza molto

elevata di rottura, rispetto alle persone che sono state trattate chirurgicamente. Una

comprensione completa dei processi patologici e biomeccanici che stanno alla base

del funzionamento e la patologia del tendine sono essenziali per una migliore diagnosi

clinica e un trattamento della patologia tendinea. Per questo motivo, uno studio

piu approfondito delle componenti del tendine e necessario per chiarire i rapporti

tra struttura e funzione dei tendini.

La struttura del tendine, composta di collagene racchiuso all’interno di una ma-

trice extracellulare (MEC) ricca di proteoglicani (PG), e la chiave per la sua efficienza

meccanica. Le fibre di collagene sono le costituenti principali del tendine costrette

a sopportare la maggior parte del carico di trazione. Si pensa che i PG siano i

mediatori dell’organizzazione dell’ultrastruttura collagenosa e facilitino la trasmis-

sione della forza lungo le fibrille discontinue di collagene. Una notevole quantita di

ricerche scientifiche sono gia state eseguite per definire l’influenza dell’ultrastruttura

sulle proprieta meccaniche del tendine. Tuttavia, la grande quantita di risultati spe-

rimentali pubblicati finora propongono conclusioni contrastanti per quanto riguarda

i dati fondamentali del comportamento ultrastrutturale del tendine. L’obiettivo di

Riassunto

questa tesi e quello di spiegare questi risultati contrastanti partendo da un approccio

“di base”, per cui vengono prese in considerazione le forze intermolecolari di base dei

legami all’interno della MEC, tra le fibrille di collagene e i PG, oltre alle proprieta

di resistenza meccanica individuali delle fibrille. Questo puo aiutare a riconciliare i

risultati contrastanti che sono stati finora osservati negli esperimenti di laboratorio,

e in ultima istanza, contribuire a confermare la diagnosi clinica per i trattamenti dei

disturbi ai tessuti connettivi.

In particolare, e stato sviluppato e validato un nuovo protocollo per testare mec-

canicamente i tendini d’Achille, al fine di quantificare il contributo meccanico della

concentrazione di PG sottoposti a un carico di trazione. I tendini sono stati trattati

chimicamente per eliminare parzialmente le catene secondarie dei PG — ovvero i

glicosamminoglicani (GAG) — e confrontati con quelli naturali. I risultati hanno

mostrato un comportamento meccanico eterogeneo, in tensione lungo l’asse longitu-

dinale, soprattutto all’inserimento del muscolo e dell’osso, evidenziando l’importanza

di eseguire un’analisi locale della deformazione rispetto alla resistenza meccanica del

tendine. Macroscopicamente, il contributo dei PG alla deformazione del tendine in

trazione meccanica ha indicato un rapporto eterogeneo e complesso tra la struttura

e la funzione dei tendini.

Inoltre, due gruppi di topi “inbred strain”, che mostrano simile comportamento

macroscopico ma hanno un diverso modulo elastico, sono stati ulteriormente esami-

nati nella loro struttura, per investigare l’influenza delle fibrille di collagene. Nuovi

strumenti di analisi d’immagine per parametrizzare la morfologia delle fibrille di

collagene sono stati progettati. Il gruppo con il piu alto modulo elastico ha esibito

strutturalmente un raggio medio delle fibrille di collagene piu grande, una superficie

fibrillare specifica (ovvero il perimetro delle fibrille in contatto con la matrice non

collagenosa) piu piccola e una minore concentrazione di GAG. Come in precedenti

studi, un maggiore raggio di fibrille di collagene sembra essere associato ad un ten-

dine rigido, ma questa differenza funzionale potrebbe anche essere attribuita alla

riduzione del potenziale di superficie di scambio tra fibrille e la matrice circostante

ricca di PG, le cui catene idrofile secondarie possono promuovere lo scivolamento

inter-fibrillare aumentando l’idratazione.

Infine, le interazioni tra i PG e le fibrille di collagene sono state studiate a li-

vello microscopico, nel loro ambiente fisiologico. Un nuovo approccio sperimentale,

che unisce il carico meccanico del tendine a livello microscopico, e stato svilup-

pato e validato, con una valutazione morfometrica delle deformazioni longitudinali

e trasversali delle fibrille di collagene a livello nanoscopico.

Le fibrille di collagene sono state caratterizzate con il microscopio a forza ato-

x

Riassunto

mica, per tendini con differenti concentrazioni di PG che sono stati sottoposti a

diversi valori di deformazione macroscopica. Il contributo meccanico dei PG a li-

vello nanoscopico e stato quantificato confrontando la differenza di lunghezza nella

caratteristica periodicita strutturale — il cosiddetto d-periodo — e nel diametro

delle fibrille tra tendini naturali e tendini impoveriti di GAG. I tendini con una

minore concentrazione di GAG hanno mostrato un maggiore allungamento nelle

fibrille di collagene.

In conclusione, riassumendo tutti gli studi specifici effettuati in questa tesi, la

concentrazione di GAG non influenza significativamente la risposta globale mec-

canica del tendine. Tuttavia, a livello microscopico, i GAG sono responsabili dei

cambia-menti nell’allungamento delle fibrille di collagene, che risulta maggiore per

una concentrazione minore di GAG. Questi ultimi sembrano quindi non legare le

fibrille di collagene al fine di trasferire meccanicamente forze tra loro in tensione,

come si credeva in precedenza, ma piuttosto idratare la MEC, e migliorare lo scorri-

mento tra le fibrille di collagene per evitare lesioni probabilmente dovute all’eccessiva

frizione.

xi

Chapter 1

Introduction

Thesis motivation

A considerable number of scientific investigations have already been performed to

define the influence of ultrastructure on the mechanical properties of tendon. How-

ever, the large body of published experimental evidence yields conflicting conclusions

with regard to the fundamental ultrastructural determinants of tendon behavior.

For example, numerous studies suggest that increased fibril diameter is primarily

responsible for increased tendon stiffness and strength [1, 2, 3, 4], while others in-

dicate that the total fibril surface area and the network of proteoglycans (PGs)

are responsible for it [5, 3]. The use of transgenic “knockout” mouse models has

permitted the focused investigation of specific ultrastructural components, such as

the proteoglycan decorin, which has historically been hypothesized to be a critical

factor in fibril organization and load sharing between adjacent fibrils. However, the

findings from these knockout studies have often been counterintuitive, implying that

fundamental assumptions regarding the manner in which fibrils are interconnected

in the extracellular matrix (ECM) must be re-evaluated.

The present study takes a “bottom up” approach, by which the functional proper-

ties of tendons are considered in conjunction with the basic intermolecular binding

forces between the collagen fibrils and PGs in the ECM. The accurate investigation

of multiscale structural properties may help reconcile the conflicting and counterin-

tuitive results that have thus far been observed in laboratory experiments, and will

ultimately help inform clinical diagnosis and treatment of connective tissue disor-

ders.

This dissertation relies heavily on the mouse as a model for human tendon struc-

ture and function. The use of the mouse as a model for human musculoskeletal

diseases has increased in popularity since the mouse genome was well character-

ized [6, 7, 8]. The advantages of using the mouse model are that various strains

have been observed to exhibit disease state characteristics similar to those found

Introduction

in humans, and that the mouse is easily accessible to manipulation of the genetic

makeup by either gene knockout, gene overexpression (transgenes), or genetic breed-

ing strategies [9]. With the exception of identical twins, the genetic background in

humans varies significantly from one individual to another, making studies of ge-

netic involvement in a given tendon phenotype difficult. Well characterized animal

lines, such as murine inbred strains are therefore used as an approach to study more

heterogeneous populations [10].

The project

The concept for this study was born from a collaboration of the Institute for Biome-

chanics within the European Union Integrated Project “Adult mesenchymal stem

cell engineering for connective tissue disorders; from the bench to the bed side”. This

project involved a consortium of 26 European laboratories, that was focused on the

use of mesenchymal stem cells (MSCs) to repair injured or pathological bone, ten-

don and cartilage. As part of this project, the Institute for Biomechanics, performed

quantitative biomechanical assessment of tendon function, and was responsible for

determining whether MSC therapies are in fact accelerating or improving the healing

process. Initial research within the EU project made it clear that more information

regarding the ultrastructural mechanics of tendon was required in order to better

direct clinical intervention strategies. While it is possible to determine whether a

particular therapy restores tendon function, the process yields little information as

to why a treatment works or not. Specifically, it was unknown whether restoration of

normal distributions of collagen fibrils should be a primary clinical goal, or whether

an increased/decreased concentration of GAGs in the ECM would more effectively

improve the biomechanical performance of healing tendon.

Specific aims

In an effort to improve novel tendon treatments, this doctoral thesis aims to investi-

gate how the individual molecular tendon components contribute to the functional

properties of tendon tissues. Tendon component influences on the mechanical behav-

ior are individually investigated through various combinations of selective chemical

digestion, microscopic observations, and tensile testing. The focus on each single

component can in fact be related with specific diseases that could occur in the hu-

man body. For instance, the behavior of GAG-depleted tendon could be associated

with the tendon of older people, in which the amount of PG naturally decreases

with age [11], or it is also found to be lower for tendon with pathologies that lead to

2

Introduction

early injuries [12]. In the work, murine tendons are observed from the macro down

to the micro- and the nano-scale to quantify structural differences at these levels of

structural hierarchy and relate them to the mechanical response.

The foci of the project can be summarized by the following three specific aims:

Specific aim 1 Investigate the functional role of proteoglycan sidechains in tensile

mechanics at the macroscale and quantify their contribution.

Specific aim 2 Define the collagen fibril morphological parameters that influence

the tendon tensile mechanical response.

Specific aim 3 Relate the interaction between the PGs and collagen fibrils in their

native network at the nanoscale with the overall mechanical response.

This work involved the development and validation of suitable experimental set-

ups, protocols, and image analysis tools to achieve these aims. The following sections

explain in more detail the outline of the thesis from the basic physiology of tendon,

the strategies for investigating the mechanical role of the tendon components, and

summarize our current understanding of the tendon structure-function relationships.

Outline of the thesis

The thesis is structured into the following five chapters: an introduction, a chapter

of general background information, targeted studies to answer open questions, and

a concluding synthesis of the work performed and outcomes achieved.

Chapter 1 is an introduction to the motivations behind the thesis. It includes

the basic information that inspires the project outline, and a listing of the specific

aims.

Chapter 2 provides the necessary background on tendon structure related to the

mechanical properties and the state of the art that frames the research performed

in the thesis.

In the following chapters the influence of the tendon components on the mechan-

ical properties are investigated with different techniques starting from the tissue-

organ level to the material level. Specifically, the following studies were carried

out:

Chapter 3 is an analysis at the tissue-organ level that mainly focuses on the

mechanical response as a correlation to different component parameters. In Chap-

ter 3.1 the investigation at the macroscopic level, of the mechanical influence of

proteoglycans on tendon behavior is presented. Specifically, this influence is corre-

lated with the PG content in the tissue. Tensile tests are performed on both natural

3

Introduction

and GAG-depleted tendon. The tendon is locally analyzed to give quantitative infor-

mation about the structural heterogeneity of the tissue (i.e. regional GAG content),

and whether any difference is reflected in the functional properties.

Chapter 4 is an analysis at the material level, of the mechanical response of

the collagen fibrils to elucidate the force transfer mechanism between them. Chap-

ter 4.1 focuses on how fiber morphology influences the mechanical response to ten-

sile load. Two genomic inbred mouse strains, showing different fiber morphology,

are compared with respect to their mechanical properties. Transmission electron mi-

croscopy (TEM) pictures of the tendon cross-section are quantitatively analyzed and

parameterized to extract information about collagen fibril diameter, fibril density,

and interface between collagen and matrix. The two groups are tested in tension

to correlate the morphology with the corresponding mechanical properties without

any chemical tissue alteration.

In Chapter 4.2 a novel imaging method needed to monitor collagen fibril de-

formation is validated and presented. This method combines the mechanical tests

performed at the macroscale and presented in the previous chapter with an atomic

force microscopy (AFM) image analysis that allows quantification of the collagen

fibril deformation while in their physiological environment. In Chapter 4.3 the

novel imaging method is implemented to investigate collagen fibril elongation occur-

ring within the tendon fascicles dependent on the PG concentration. Specifically,

changes in the collagen fibril elongation in relation to the PG concentration are

analyzed with AFM. Two groups of tendons are chemically fixed in their relaxed

position and at increasing target strains. Images are taken for both natural and

GAG-depleted tendon. The measured fibril elongation for equal applied-strain is

correlated to GAG content and compared.

In Chapter 5 the thesis concludes with a synthesis of the key findings, a discussion

that combines all the results and compares them with previous studies, and possible

future work.

References

[1] D. A. Parry, “The molecular and fibrillar structure of collagen and its relation-

ship to the mechanical properties of connective tissue,” Biophys Chem, vol. 29,

no. 1-2, pp. 195–209, 1988.

[2] J. Liao and I. Vesely, “A structural basis for the size-related mechanical proper-

ties of mitral valve chordae tendineae,” J Biomech, vol. 36, no. 8, pp. 1125–33,

2003.

4

References

[3] T. C. Battaglia, R. T. Clark, A. Chhabra, V. Gaschen, E. B. Hunziker, and

B. Mikic, “Ultrastructural determinants of murine achilles tendon strength dur-

ing healing,” Connect Tissue Res, vol. 44, no. 5, pp. 218–24, 2003.

[4] C. Y. Lin, N. Kikuchi, and S. J. Hollister, “A novel method for biomaterial scaf-

fold internal architecture design to match bone elastic properties with desired

porosity,” J Biomech, vol. 37, no. 5, pp. 623–36, 2004.

[5] B. Mikic, B. J. Schalet, R. T. Clark, V. Gaschen, and E. B. Hunziker, “Gdf-5

deficiency in mice alters the ultrastructure, mechanical properties and compo-

sition of the achilles tendon,” J Orthop Res, vol. 19, no. 3, pp. 365–71, 2001.

[6] N. G. Copeland, N. A. Jenkins, D. J. Gilbert, J. T. Eppig, L. J. Maltais, J. C.

Miller, W. F. Dietrich, A. Weaver, S. E. Lincoln, R. G. Steen, and et al., “A

genetic linkage map of the mouse: current applications and future prospects,”

Science, vol. 262, no. 5130, pp. 57–66, 1993.

[7] R. P. Erickson, “Why isn’t a mouse more like a man?,” Trends Genet, vol. 5,

no. 1, pp. 1–3, 1989.

[8] J. H. Nadeau and B. A. Taylor, “Lengths of chromosomal segments conserved

since divergence of man and mouse,” Proc Natl Acad Sci U S A, vol. 81, no. 3,

pp. 814–8, 1984.

[9] L. Silver, Mouse Genetics. New York: Oxford University Press Inc., 1995.

Thomas.

[10] R. Voide, G. H. van Lenthe, and R. Muller, “Differential effects of bone struc-

tural and material properties on bone competence in c57bl/6 and c3h/he inbred

strains of mice,” Calcif Tissue Int, vol. 83, no. 1, pp. 61–9, 2008.

[11] E. H. Stephens, C. K. Chu, and K. J. Grande-Allen, “Valve proteoglycan con-

tent and glycosaminoglycan fine structure are unique to microstructure, me-

chanical load and age: Relevance to an age-specific tissue-engineered heart

valve,” Acta Biomater, 2008.

[12] G. P. Riley, R. L. Harrall, C. R. Constant, M. D. Chard, T. E. Cawston, and

B. L. Hazleman, “Glycosaminoglycans of human rotator cuff tendons: changes

with age and in chronic rotator cuff tendinitis,” Ann Rheum Dis, vol. 53, no. 6,

pp. 367–76, 1994.

5

Chapter 2

Background

2.1 An analogue for tendon

2.1 An analogue for tendon

2.1.1 Structure function

In everyday life objects or materials that are used are chosen for their function,

which is defined by their structure. The same construction materials can be used

to build different structures like bridges or skyscrapers that have a different func-

tion. Stepping further into the structure, from a general perspective, all materials

are made of atoms. Depending on its atomic structure, the same atom can form

materials with totally different functions as in the case of diamond and graphite,

shown in Figure 2.1.

Figure 2.1: Function related to the structure of the same ground material. Thecarbon atom with a different atomic structure is the ground material oftwo totally different materials like diamond and graphite.

This simple example shows how the structure and function of a material are

related. Even if all the components of a material are known, the structure is critical

to understand and predict its function. This study will focus on the structure at

the microscale of a tendon, whose structure and function can be compared with a

rope, as explained in the following section.

9

Background

2.1.2 The rope

Ropes are used to bind together many things, connect two objects to their ends

with a knot or as a security for sports, like mountaineering or climbing. Some of

their functions include attaching, carrying, fastening or lifting. In all of these tasks,

ropes are subjected to tensile load. The key to the performance of ropes in tension

is in their unique structure: a core of fibers, which are twisted or braided together

at different levels to provide elasticity; the core is further covered with an additional

layer of intertwined fibers to protect them from local damage due to cuts or friction

(see Figure 2.2).

Figure 2.2: Schematic section of a rope.

Similar to ropes, tendons attach bones to muscles in the human body to “carry”

the force produced by the muscle to “lift” the bone. Their performance is also a

feature of their structure composed of fibers grouped together at different levels to

give elasticity and strength, embedded into a sheath that allows the tendon to glide

and protect the core from friction.

Tendon and rope structures differ in two main aspects: tendons cannot be tied

at their ends but consist of a single structure that binds two different tissues (the

muscle and the bone); and tendon fibers — for example, those in the Achilles tendon

— are parallel to each other rather than twisted or braided. These apparently

minor differences in the structure are mechanically challenging. The challenge is

in providing a continuous structural transition between a soft elastic muscle to a

hard stiff bone without losing energy during locomotion. For this reason a lot of

interest is being directed across the research world into the tendon structure-function

relationship that is discussed in the following section.

10

2.2 Tendon function

2.2 Tendon function

2.2.1 Anatomical role

The tendon’s main role is to transmit muscle generated force to a bone and enable

locomotion [1]. In the human body there are two kinds of tendon: positional tendons

and energy storing tendons. Positional tendons principally transmit force to bone

to move joints and they are relatively inextensible under physiological loads. The

energy-storing tendons also act like a spring to store elastic energy and increase the

efficiency of locomotion [2] and are required to stretch and recoil under physiological

loads to ensure return of stored energy [3]. The difference between these two tendon

groups is also reflected in the mechanical properties. The energy-storing tendons

show a significantly lower elastic modulus (E-modulus) than the positional tendons,

in order to support optimization for their specific physiological roles [4].

The Achilles tendons is an example of an energy-storage tendon [5], and it will

be the main subject of this study. A detailed description of its mechanical behavior

under tensile load will be described in the following section.

2.2.2 Tensile behavior

Tendon under tensile load presents a typical stress-strain curve that can be explained

by three different mechanical behaviors summarized in three main regions displayed

in Figure 2.3: the toe region, the heel region and the elastic region. In the toe

region, the macroscopic “crimp” formed by the fascicle disappears, while there are

no further structural changes visible with the light microscope at larger strains.

This crimp pattern of the tendon fascicle is believed to play a major role in the

mechanical behavior of tendon, specifically for small loads that correspond to the

toe region [6, 7]. It has been suggested that the crimp provides a shock dissipation

mechanism that is evident in the compliant toe region of the load-deformation curve

[7, 8]. Local variations in tissue stress have been associated with region-specific

differences in crimp angle [9, 10]. It has also been shown that patients clinically

presenting a rupture of the Achilles tendon possess significant differences in collagen

fibril diameter distribution and crimp when compared to non-ruptured controls [11].

In the heel region an increase in the E-modulus is observed. Some studies have

found that as in the fascicles, at the fibril level there are also micro crimps, so-called

kinks [12, 13]. A change in the E-modulus in the heel region is therefore believed

to be a change in the intermolecular spacing or in the degree of order in the fibril

gap regions. X-ray studies reported no net reduction in the intermolecular spacing

11

Background

within the fibrils but a dramatic reduction in the entropic disorder under tension

[13, 14, 6]. In other words the kinks present in the fibril’s gap straighten with

applied load before any significant elongation is observed in the fibrils themselves

[12, 13, 15]. This mechanism is basically a form of rubber elasticity, due to a further

loss of freedom of motion of the gap zone segments between the collagen molecules.

In the elastic region the whole tendon structure is already under tension and exhibits

a linear mechanical response in relation to the applied load.

Figure 2.3: Typical stress-strain curve and corresponding images at the macroscale(visualized using polarized light) and microscale (structural changes oc-curring at the fibrillar level) of a rat tail fascicle. In the toe region themacroscopic crimps disappear, in the heel region the molecular kinksin the gaps regions progressively straighten, while in the elastic regionthe intermolecular spaces elongate and the fibril order increases. Figureadapted from Fratzl [6].

12

2.3 Tendon structure

2.3 Tendon structure

Tendons are made of groups of fascicles that are connected to the muscles. Each mus-

cle ends in a different fascicle that can have a different orientation in the structure

depending on the bone to which it is attached. Specifically, tendon has a multi-level

hierarchical structure composed of tendon units, fascicles, fiber bundles, fibrils and

collagen molecules that run parallel to the tendon’s long axis (Figure 2.4).

Figure 2.4: Organization of tendon as identified from nanoscale imaging techniques(x-ray diffraction, electron microscopy) to microscale (scanning electronmicroscopy, visible light microscopy). This classic figure is adapted fromKastelic [16].

The fascicles are wrapped by a thin walled sheath: the endotendon. The low

friction coefficient of the endotendon allows the independent relative movement of

the fascicles within the tendon [17]. Each fascicle presents the same structural

characteristics showing a “crimp” pattern along the longitudinal axis. Crimping

can be observed as a periodic banding of dark lines using polar light microscopy

(Figure 2.3), and have been quantified in several studies [18, 19, 14] as a period

ranging from 10 to 20 µm, with an amplitude of 3 to 4 µm, and an angle ranging

between 30 to 40 degrees. Their function is to give elasticity from the unloaded

state to the elongated state without losing tensile strength [6]. Within the fascicle,

the collagen fibrils embedded in a proteoglycan-rich matrix form the extracellular

matrix (ECM).

13

Background

2.3.1 Collagen

Collagen is the most abundant protein in mammals. About one quarter of the

protein in the body is collagen, which is also the main component of all connective

tissues [20]. Collagen provides structure to our bodies, protecting and supporting

the softer tissues and connecting them with the skeleton. There are several types of

collagen present in the human body as shown in Table 2.1.

Table 2.1: Collagen types present in the body.

Type Polymerized form Tissue distribution

Fibril forming I fibril bone, skin, tendons, ligaments, cornea,

internal organs

II fibril cartilage, intervertebral disc, noto-

chord, vitreous humor of the eye

III fibril skin, blood vessels, internal organs

V fibril associated with type I

XI fibril associated with type II

Fibril associated IX lateral association

with type II

cartilage

XII lateral association

with some type I

tendon, ligaments, some other tissues

Type I collagen constitutes around 60% of the dry mass of tendon and 95% of the

total collagen [21]. Fibrils are easily recognizable by their characteristic, so-called, d-

Figure 2.5: Schema and micrographs of the d-period. The schema on the left showsthe d-period regular stripe visible on the collagen fibrils surface along thelongitudinal axis. On the right side, experimental view of the d-periodwith different imaging techniques: atomic force microscope (AFM), scan-ning electron microscopy (SEM) and transmission electron microscopy(TEM).

14

2.3 Tendon structure

period, i.e. a regular stripe on the fibrils surface along the longitudinal axis as showed

in Figure 2.5. This d-period is formed by the peculiar axial packing of the collagen

molecules, as illustrated in Figure 2.6. First the cell secretes a procollagen that is

converted to collagen by removal of the propeptide ends with enzymes. Lateral forces

induce the collagen molecules to spontaneously self-assemble into fibrils. Eventually,

the fibrils are stabilized by covalent cross-linking initiated by enzymes (lysil-oxidase)

[22]. The structural characteristic of the fibrils is explained by the Hodge-Petruska

model as a result of the molecule staggering [23].

Figure 2.6: Formation and self-assembly of collagen fibrils. Procollagen is secretedfrom the cell and cleaved at the propeptide ends to form a collagenmolecule. The fibril is formed by the spontaneous parallel self-assemblyof the collagen molecules. As a last step the fibrils are stabilized bycovalent cross-linking, which is initiated by lysil oxidase.

The length of a collagen molecule (∼ 280 nm) was recognized to be 4.4 times

greater than this characteristic d-period of 67 nm. This discrepancy between the col-

lagen molecule’s length and arrangement suggests the concept of a quarter-stagger,

consisting of overlap and gaps between the molecules as shown in Figure 2.7. The

15

Background

collagen molecules spontaneously assemble laterally with the same configuration

forming overlapping and low density stripes in the fibril that exhibits a regular

periodicity.

Figure 2.7: Schematic representation of a tendon fibril. The spontaneous lateral ar-rangement of the collagen molecules results in an overlap of the molecules(labeled with O). The gap region (labeled with G) appears in onemolecule out of five and results in the d-period. Figure is adapted fromHodge-Petruska [23].

2.3.2 Proteoglycans

As mentioned previously, collagen fibrils are embedded in the ECM, which contains

another important constituent: the proteoglycans (PGs). PGs have a core protein

to which one or more glycosaminoglycan (GAG) side chains are covalently bonded.

In Figure 2.8 a schematic of a PG aggregate, to which several GAGs such as der-

matan sulfate (DS), chondroitin sulfate (CS) and heparan sulfate (HS) are covalently

bonded. Various types of PG are present in the tendon and they constitute 1% to

5% of the tendon dry mass. One of the so-called small leucine rich PGs, decorin, is

widely distributed in the tendon and it is known to modulate the formation and the

final sizes of the fibrils in the ECM [24, 25, 26, 27, 28, 29]. In the tensile region of

the tendon, decorin is the predominant PG, while in the region where tendon inserts

or wraps around bone both small leucine rich PGs —decorin and biglycan— and

larger PGs like aggrecan are present [25, 30, 31]. Decorin has only one GAG chain

of either chondroitin or dermatan sulfate. DS constitutes 60% of the GAG mass in

the tensile area and CS constitutes 65% of the GAG mass in the bone insertion zone

[30].

GAGs are unbranched polysaccharide chains, which are strongly hydrophilic.

Their high density of negative charge attracts sodium cations (Na+) present in the

16

2.4 Structure-function models

core protein core

protein

GAG100 nm

Aggrecan (MW ~ 3 x 106) Decorin (MW ~ 40000)

Figure 2.8: Schema of a proteoglycan aggrecan and decorin. The aggrecan is a largeprotein, to which several glycosaminoglycans are covalently connectedto the core protein, while the decorin is the smallest PG and has onlyone GAG chain. Adapted from Alberts et al [32].

tissue, that osmotically attract a large amount of water in the matrix. The osmosis

creates a swelling pressure that enables the matrix to bear compressive forces as in

the case of the cartilage where the collagen network traps the PG chains and holds

them “in place” in the ECM [33]. Thus, PGs keep the matrix hydrated [34, 35, 36].

The composition and organization of the PG-rich matrix is believed to influence the

sliding of the collagen fibrils [37, 38] and be directly implicated in the viscoelastic

behavior of the tendon [39].

In the ECM Scott, [40] suggested that there are interactions between PGs and

collagen fibrils. Later on Ruggeri [41] proposed that PGs are like side-chains that

connect the collagen fibrils enabling transfer forces between them. Decorin and its

DS and CS chains, being the main PGs found in tendons, have been described by

several authors as being directly implicated in the mechanical integrity of tendon

[42, 43, 44, 13, 45], and have since became the main subject of study for models

trying to explain the tendon mechanical response.

2.4 Structure-function models

2.4.1 Composite material theory

The relationship between the distribution of collagen fibrils and functional behavior

of tendon has been investigated in numerous experimental studies. Several of these

suggest that increased fibril diameter is a primary determinant of improved tendon

stiffness and strength [46, 18, 47, 48]. More recent studies have shown however,

that despite a generally small fibril diameter, healing tendon gains strength with an

increasing degree of fibril area fraction (Figure 2.9d), and that factors apart from

fibril diameter may be critical to improved tendon strength [47]. Consistent with this

concept, a different study indicated that Achilles tendons of Growth/Differentiation

17

Background

Figure 2.9: Cross-sectional histological slide of (left) healthy control mouse Achillestendon, and (right) healing Achilles tendon at 42 days post-operation.The bar indicates approximately 125 nm [47].

Factor 5 (GDF5) deficient mice showed drastically reduced functional performance

while being composed of nearly normal fibril diameter distributions and fibril area

fractions [49]. Generally these studies indicate that biochemical deficiencies in the

non-collagenous ECM may be a primary causative factor in certain tendon patholo-

gies.

If the tendon is considered to be a composite material made of stiff fibers em-

bedded in softer matrix, the E-modulus of the material can be calculated with the

following equation:

E-modulus of a composite material:

E = EfSf

S+ Em

Sm

S(2.1)

and S = Sf + Sm (2.2)

where Ef is the E-modulus of the fibers, Em is the E-modulus of the matrix, Sf

is the surface of the fiber, Sm is the surface of the matrix and S the total area of

the material. Isolated PGs are known to form a gel-like substance with a shear

stiffness of about 0.05 kPa. This is eight orders of magnitude lower than the tensile

modulus of collagen (2.9 ± 0.1 GPa) [50], therefore Ef >> Em. If this equation

18

2.4 Structure-function models

can be applied to calculate the E-modulus of a tendon, the collagen density should

be the main source that influences its stiffness. If this is true the collagen fibril

diameter without information on the fibril fraction area is not a sufficient parameter

to determine tendon strength.

In fact, the mechanical properties of tendon are based not only on the orientation,

density, and length of the collagen fibrils, but also on intra- and inter-molecular cross-

links between other components of the ECM and the collagen fibrils [51, 50, 52, 53,

54]. The biochemical makeup of the ECM influences the functional mechanical

properties including the viscoelastic characteristics of the tissue and its response

to impulsive loading [9, 55]. Molecular cross-linking in connective tissue involves

a complex enzymatic interplay between the ECM components during growth and

maturation, and is regulated, at least in part, by physical activity [56, 57, 58, 59, 60].

Based on experimental analysis, studies support the idea that PGs connect the

fibrils to transfer load [24, 41, 43, 42, 27]. After load application, a relative move-

ment between fibrils and a corresponding change in the fibril-PG configuration was

observed. Specifically, Liao showed a skewness angle of interfibrillar PGs that in-

creased with applied load [43].

2.4.2 Shear-lag theory

The shear-lag theory assumes that there are perfect connections between the fibers

and the matrix, and that both fibers and matrix show an elastic behavior. Thus, the

shear forces between adjacent fibers, distributed uniformly along the entire length

of the fiber, transfer the load into the surrounding matrix [61, 62, 63]. Connective

tissue modeling [64] has introduced this concept in an effort to illuminate the role

of interstitial GAGs. Thus, consistent with this theory, the cumulative effect of

these weak forces could highly influence the mechanical strength of the tissue. The

single fibril tensile contribution in the network would be then determined by the

efficiency of the shear forces transmission between adjacent fibrils through PGs,

which is related to their concentration in the structure (see figure 2.10).

Theoretical models have also been introduced based on this theory. These mod-

els quantified the binding force of decorin to the collagen fibril [45], or the force

transferred between fibrils [44] in the range of some micronewtons. If we considered

tendons as composite materials composed of stiff fibers surrounded by a softer ma-

trix, the cumulative effect of these weak forces could highly influence the mechanical

strength of the tissue.

19

Background

Figure 2.10: Schema of the similar function in tension of a tendon and a compositematerial. The tendon structure is composed of collagen fibrils connectedwith PGs within the ECM and the composite material is composed of astiff fiber embedded in a softer matrix that follow the shear-lag theory.

2.4.3 Open question

While the actual contribution of PGs to tensile tendon mechanics remains largely

undefined, a simplified estimate of the PG linkage density in a single tendon fascicle

gives an understanding of the potentially important role played by PG interconnec-

tions. Since tendon fibrils have an average fibril diameter of approximately 50 nm

and a d-period of 67 nm, one d-period of a single fibril can be held in a cubic volume

of 50 nm × 50 nm × 67 nm. In a cylindrical fascicle with a diameter of 0.5 mm

and a length of 10 mm, and assuming that half of the volume is occupied by fibrils

and that each d-period corresponds to a single PG, there would be approximately 6

trillion PG linkages within a single tendon fascicle. Thus even with extremely small

intermolecular forces, the capacity for a substantial cumulative effect exists.

Concerning the Achilles tendon, other studies have shown that there is a difference

in GAG concentration between the tensile and the insertion regions of the tendon.

Specifically, the tensile region is reflected with a larger abundance of the small

PGs such as decorin, and the compressed region have a more fibrocartilaginous

phenotype with large PGs of aggrecans [4]. This results in a concentration of GAGs

in the insertion region that can be up to 25 times higher than in the midsubstance

[30, 65, 66, 67]. This implies that the mechanical properties along the tendon might

be mediated by GAG content, or may simply reflect compressive loading at the

20

References

bone.

Recent conflicting studies, comparing native and GAG-depleted tendon groups,

challenge this theory. Indeed no evidence of the PG contribution to the tensile

properties of the tissue were proved [68, 69, 70]. Both studies found no influence

related to the concentration of PG to the mechanical response of soft tissue at the

macroscale. It thus remains unclear whether or not the increase in the skewness angle

of interfibrillar PGs with applied load is a conformational change in PG arrangement

under stress.

These results confuse the question of how force transmission between components

occurs. Some support the idea that PGs transfer force between fibrils [40, 44] while

others that they rather influence the sliding behavior of the fibril [37, 71]. Another

open question is whether the major source of the tendon stiffness is regulated by

the collagen fibrils [46, 72], or the PG network [51, 49]. Although the mechanical

properties and structure of tendon are well characterized, it remains unclear how

the structure and organization of the tendon leads to its properties. In the following

chapters, the tendon structure and its related functions will be analyzed from the

tendon macroscopic level (1 - 10 mm), progressing to the fibril level (50 - 400 nm)

in order to elucidate these questions.

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28

Chapter 3

Tissue-organ level

3.1 Local strain measurement

3.1 Local strain measurement reveals a varied

regional dependence of tensile tendon mechanics

on glycosaminoglycan content

S. Rigozzi, R. Muller and J. G. Snedeker

Institute for Biomedical Engineering, University and ETH Zurich, Zurich, Switzer-

land

published in:

Journal of Biomechanics

vol(42):1547-1552, 2009

Abstract:

Proteoglycans (PG) and their associated glycosaminoglycan (GAG) side-chains are

known to play a key role in the bearing of compressive loads in cartilage and other

skeletal connective tissues. In tendons and connective tissues that are primarily

loaded in tension, the influence of proteoglycans on mechanical behavior is debated

due to conflicting experimental evidence that alternately supports or controverts a

functional role of proteoglycans in bearing tensile load. In this study we sought

to better reconcile these conflicting data by investigating the possibility that GAG

content is differentially related to tensile tendon mechanics depending upon the

anatomical subregion one considers. To test this hypothesis, we quantified the me-

chanical consequences of proteoglycan disruption within specific tendon anatomical

subregions using an optical-mechanical measurement approach.

Achilles tendons from adult mice were treated with chondroitinase ABC to

obtain two groups consisting of native tendons and GAG depleted tendons. All the

tendons were mechanically tested and imaged with high-resolution digital video

in order to optically quantify tendon strains. Tendon surface strains were locally

analyzed in three main subregions: the central midsubstance, and the proximal and

distal midsubstance near the muscle and bone insertions respectively. Upon GAG

digestion, the tendon midsubstance softened appreciably near the bone insertion,

while elastic modulus in the central and proximal thirds was unchanged. Thus the

contribution of PGs to tensile tendon mechanics is not straightforward and points

31

Tissue-organ level

to a heterogeneous and complex structure-function relationship in tendon. This

study further highlights the importance of performing local strain analysis with

regard to tensile tendon mechanics.

Keywords:

Tendon, glycosaminoglycans, mechanical properties, local strain measurement, ten-

sile testing

Introduction

Tendon is composed mainly of type I collagen embedded in a proteoglycan (PG)-rich

extracellular matrix (ECM). Type I collagen constitutes around 60% of the dry mass

of tendon and 95% of the total collagen. The fibrils are discontinuous segments that

do not run the length of the tendon [1] implying that collagen fibrils must transfer

forces to neighboring fibrils to be mechanically viable [2]. In stretched tendon, the

overall tissue strain is larger than the strain in the individual fibrils, indicating that

relative fibril movements within the matrix must take place [3, 4].

Aside from collagen, another important constituent of tendon ECM are PGs

and their associated glycosaminoglycan (GAG) side-chains such as dermatan sul-

fate (DS), chondroitin sulfate (CS), or keratan sulfate (KS). PGs constitute 1% to

5% of tendon dry mass. One of these, decorin, is widely distributed in the tendon

and is known to modulate the formation and final sizes of the fibrils [1, 5, 6, 7, 8].

Decorin is the predominant PG in the tensile region of tendon, while in the region

where tendon inserts or wraps around bone other PGs like biglycan and aggrecan are

also present [9, 10, 7]. Decorin has only one GAG, either CS or DS. DS constitutes

60% of the GAG mass in the tensile area and CS constitutes 65% of the GAG mass

in the bone insertion zone [9].

Decorin and its sulfated GAG chain have been directly implicated in the me-

chanical integrity of tendon. In the tendon ultrastructural model first proposed by

Ruggeri and Benazzo [11], decorin bonds are believed to interconnect neighboring

collagen fibrils in order to transfer forces between them. There has been further

evidence to support this model [2, 12, 13]. Conversely, some recent studies have

indicated that this model may not hold true [14, 15].

In the current study we attempt to examine the functional role, if any, of GAGs

in the tensile mechanics of the murine Achilles tendon. Our hypothesis was that

DS/CS content would correspond to tendon elastic modulus due to GAG cross-

linking of the collagen fibrils. We further hypothesized that regional variations

32

3.1 Local strain measurement

in tendon elastic modulus are partly due to relative differences in the anatomical

distribution of GAGs. To investigate these hypotheses a method to measure local

tendon strains was developed and validated, and then applied to investigate the

mechanical repercussions of targeted enzymatic digestion of DS/CS.

Materials and Methods

Tendon dissection and preparation

Achilles tendons were harvested from female 19-week-old C57BL/6 inbred (wild

type) mice reviewed and approved for use by the local authorities for all levels of

investigation. This mouse model was selected because inbred mice present genetic

homogeneity and consequently low skeletal variability [16]. Forty tendons from 20

mice were dissected with the calcaneous and the gastrocnemius/soleus muscles in-

tact. The tendon sheaths were also maintained in order to preserve the natural

anatomical structure and relative orientation of the individual tendon bundles (crit-

ical for reliable mechanical testing, unpublished data). Muscle fibers were then

carefully removed to expose the intramuscular tendon fibers.

Tendons from both legs were randomly allocated to the experimental groups. All

specimens were treated as independent samples since previous work in our laboratory

has shown variance between contralateral sides to be similar to variance between

animals (unpublished data). A control group of 20 tendons was incubated for 12 h

at 37◦C in 6ml of Tris Hydrochloride 0.1M Sodium Acetate 0.1M buffer (pH 8). A

treated group of 20 tendons was incubated for 12 h in 6ml Tris buffer + 0.15U/ml

chondroitinase ABC (C2905, Sigma-Aldrich, Switzerland), pH 8, in order to partially

digest the GAG [17].

All tendons were then rinsed in phosphate buffered saline solution (PBS) and

briefly soaked in a PBS / indigo ink mixture. By this method small regions of the

tendon surface were “permanently” stained (in an apparently random fashion), thus

providing markers that facilitated optical strain analysis. Specimens were prepared

for tensile testing by bonding the intramuscular fibers to paper with cyanoacrylate

adhesive, leaving approximately 5 mm of exposed tendon midsubstance between the

calcaneous and the bound intramuscular fibers [18]. After mechanical testing, the

tendon midsubstance was dissected proximally at the clamp interface and distally

near the bone insertion, wrapped in saline soaked gauze and immediately frozen at

-18◦C for eventual analysis of GAG content.

The frozen tendon was later thawed, carefully cut into thirds, briefly soaked in

PBS (approximately 5 seconds), blotted lightly with a paper towel, and immediately

33

Tissue-organ level

weighed. All tendon wet weight was determined using the same protocol. Addition-

ally, to define the relationship between wet weight and dry weight, three tendons

were first wet-weighed as above and then weighed after lyophilization. Dry weight

corresponded to 40 ± 4% of the wet weight. This is consistent with the literature

[15, 19] and indicated good reproducibility of the wet weighing protocol.

After weighing, the tendon subregions were digested for 16h with 500U/ml papain

solution in buffer (0.1M disodium hydrogen phosphate, 0.01M EDTA disodium salt,

14.4mM L-cysteine) at 60◦C. GAG content was determined spectrophotometrically

(Cary 50, Varian, Zug, Switzerland) at 525 nm following binding to dimethylmethy-

lene blue dye (DMMB) [20] using CS as standard. DMMB binds to negatively-

charged sulfated groups, such as CS/DS or KS.

Biomechanical testing

Mechanical tests were performed with a universal testing machine (Zwick 1456,

Ulm, Germany). Specimens were clamped for testing using a previously described

technique that minimizes failure at the clamping interfaces [18], with the calcaneous

mounted at 30◦ dorsiflexion to approximate a neutral anatomical position. Tendons

were tested in saline at 37◦C using controlled displacement (10 mm/min). Tendons

were preconditioned with 10 cycles of 0-10% nominal strain before ramp loading to

failure. Non-contact measurements of tendon cross sectional area were obtained by

frontal and lateral imaging of each tendon at a preload of 0.1 N with telecentric lenses

(Zeiss Visionmes 16.1, Zeiss, Jena Germany, pixel resolution ∼ 0.015 mm). Cross-

sectional area was determined from these measurements at the smaller measured

diameter by assuming an elliptical cross-section. Force was then normalized by the

cross-sectional area to yield nominal stress values.

Tendon strains were measured using both relative changes in clamp-to-clamp dis-

tance (i.e. “machine-based strain”) as well as optically measured strains in the ten-

don midsubstance as described in the following section. Figure 3.1 shows a param-

eterized material curve obtained from a typical tensile test (shown with machine-

based strains). These were analyzed for ultimate stress, failure strain (strain at

ultimate stress), and elastic modulus (slope of the best fit line to the “linear region”

of the material curve, as defined below).

Local strain analysis

High-speed digital video (VT Cam, AOS Technologies AG, Switzerland) was

recorded to quantify the engineering strain in the anatomical subregions illustrated

in Figure 3.2. Two frames were selected corresponding to the beginning and end of

34

3.1 Local strain measurement

Figure 3.1: Typical stress-strain curve for an Achilles tendon during uniaxial tensiletesting (left). Optical strains were extracted from video frames corre-sponding to points A and B indicated in the linear region of the testcurve. A is the end of the toe region, defined at 0.8 N; B correspondsto 80% of the ultimate load. Displacement markers (highlighted herefor emphasis) were automatically detected according to regions of highimage contrast.

the linear region of the test curve (Figure 3.1). The first frame (A) corresponded

to tendon loading at 0.8 N (approximately the end of the “toe” region), while the

last frame (B) corresponded to 80% of the ultimate load — a measure that reliably

marked the end of the linear tendon response.

A semi-automatic script was written to quantify surface deformation and corre-

sponding surface strains. A minimum of 13 displacement markers were chosen on

the surface, with all markers visible in both frames. After mapping these points of

interest, the program connected all displacement markers with a line segment. Fol-

lowing loading, the resulting elongation of each marker segment was decomposed to

determine the engineering strain component along the functional axis of the loaded

tendon. In order to minimize pixel rounding errors, only marker pairs with a min-

imum separation of 30 pixels were considered (1 pixel ∼ 0.015 mm). Further, only

vector angles that did not deviate more than 30◦ from the tendon functional axis

were considered.

The accuracy and precision of the semi-automated local strain analysis was in-

vestigated using an isotropic rubber test phantom submitted to the tendon test

protocol. Optically derived local strains were highly consistent with structural val-

ues obtained from the testing machine, and gave accurate and precise local strain

values for all considered marker pairs with less than 5% measurement error. The

35

Tissue-organ level

Figure 3.2: Posterior view of a murine Achilles tendon clamped for tensile testing.The optical strain was calculated using video of the visible tendon fibers,while machine-based strain was automatically determined from the ma-chine clamp to clamp distance. For a focused local strain analysis, thetendon midsubstance was evenly divided into three parts, the proximalregion included a portion of the tendon to the intramuscular fibers, thecentral region comprised the middle third of the tendon midsubstance,and the distal region included the transition from midsubstance to thetendon-bone insertion.

local strain algorithm was also submitted to a repeatability study, and indicated

an absolute error lower than 2% for independently analyzed trials (same user) of

individual force-video datasets.

Statistical analysis

A Lilliefors test was used to determine whether the data distribution was normal.

Normally distributed data were compared using a pair-wise t-test with Holm cor-

rection for multiple comparisons. A pair-wise Wilcoxon rank sum test was used to

compare groups with non-parametric distribution. Relationships between the two

groups were computed with single and multiple linear regression analyses. A p-value

< 0.05 was considered to be significant.

36

3.1 Local strain measurement

Results

GAG Digestion

The GAG assay showed that in the control group there was a statistically significant

difference in GAG concentration between the subregions (Figure 3.3). Chondroiti-

nase ABC treatment most affected the middle subregion, which lost 54% of GAG

content. The proximal and distal subregions were digested of 47% and 44% of the

total amount respectively. After digestion, the remaining GAG concentration was

found to be similar in all tendon subregions.

Figure 3.3: Chondroitin sulfate weight per wet tendon weight (µm/mg) for the threemidsubstance regions as determined by GAG assay. The relative sub-region contribution to total midsubstance GAG content is also shownas a percentage. All columns connected by a star (?) are statisticallydifferent at p < 0.05.

Biomechanical testing

Two samples from each per group were excluded for cases in which a calcaneous

avulsion or intramuscular tendon fiber tear were observed. The others failed in

the midsubstance. Video analysis revealed no slippage at the clamp. In general,

the natural tendons demonstrated a sudden failure of a twisted bundle that was

37

Tissue-organ level

visible through the sheath. For the digested group, the rupture was less dramatic,

and did not obviously involve fiber bundle torsion. A mean cross-sectional area of

0.58 ± 0.14 mm2 was measured for the control group, while for the digested group

it was 0.48 ± 0.10 mm2 (p < 0.05).

Table 3.1: Summary of the measured parameters.

Machine-based values

Groups L0 (mm) Cross-

sectional

area (mm2)

Ultimate

force (N)

Ultimate

stress

(MPa)

Strain at

failure (%)

Stiffness

(N/mm)

Elastic

modulus

(MPa)

Control 5.5±0.7 0.58±0.14* 6.4±2.1* 11.1±4.9 36±17 33.8±20.8 61.8±38.8

Digested 5.2±0.9 0.48±0.10* 5.0±1.8* 11.5±5.6 36±19 25.4±19.8 57.9±45.9

Values are expressed as mean ± standard deviation. * Statistical significant for p < 0.05

Effects of GAG digestion on tensile tendon mechanics

Measurements of tendon modulus and failure behaviors satisfied tests of fit to a nor-

mal distribution and were analyzed using Students t-test to compare group means.

Optical measurements of midsubstance tendon strains were not normally distributed

according to Lilliefors test, and were thus analyzed using appropriate parametric sta-

tistical tests. As summarized in Table 3.1, a 50% digestion of GAGs over the entire

tendon was associated with a significant decrease in ultimate load, but with no sta-

tistical change in ultimate tensile stress, failure strain, stiffness, or elastic modulus

when strains were normalized to machine gage length (clamp to clamp distance).

Conversely, normalization of data using optical strain measurements of the tendon

Table 3.2: Elastic modulus values for the tendon midsubstance and its three com-ponent subregions, based on local strain measurements.

Modulus MPa

Midsubstance Proximal third Central third Distal third

Control 117 (50;225)* 120 (57;221) 92 (45;195) 131 (66;242)*

Digested 67 (30;117)* 78 (34;214) 114 (39;245) 69 (21;117)*

p-value <0.05 1.000 1.000 <0.001

Values are expressed as median (first percentile; third percentile). * Denotes a significant p-value at the indicated

level.

midsubstance indicated a significant decrease of 46% in elastic modulus after diges-

tion (p < 0.05, Table 3.2). In both control and digested tendons, the optically-based

38

3.1 Local strain measurement

Figure 3.4: (A) Median stress vs strain curves for control and GAG digested murineAchilles tendon. After digestion, the GAG-depleted group (dashed gray)shows a lower modulus than the control group (black). (B) The regres-sion line indicates the relationship between the machine-based elasticmodulus and the optically-based elastic modulus for both the controland the digested groups.

elastic modulus was nearly twice that of the machine-based modulus. The median

stress vs. strain curves for both control and digested tendon as well as the correlation

between optically-based and machine-based moduli are summarized in Figure 3.4.

Figure 3.5: Box plot of (A) the strain and (B) the elastic modulus in loaded tendon,as measured in the proximal, central and distal region of the tendonmidsubstance. The distal subregion of GAG-depleted tendon was statis-tically different from corresponding controls with p-value < 0.05.

39

Tissue-organ level

A more localized analysis of the midsubstance subregions revealed non-homogeneous

distributions of strains in the tendon (Figure 3.5A). Enzyme digestion differently af-

fected the more proximal subregions of the midsubtance when compared to the most

distal third. Specifically, GAG digestion did not affect the strain distributions within

the proximal and central midsubstance but significantly increased the tendon strains

closer to the bone insertion. Thus the observed 46% decrease in tendon modulus

associated with GAG removal was attributable to decreased modulus towards the

bone insertion rather than over the entire tendon (Figure 3.5B).

Discussion

In this paper we have investigated the mechanical role of GAGs in tendon under

tensile load. Local mechanical properties were correlated to local GAG content in

distinct subregions of the tendon. It was shown that partial removal of CS/DS

does not alter distribution of strains in the proximal and middle portions of the

midsubstance, but does have a significant effect at the distal region closer to the

bone insertion.

While localized analysis of tendon optical strains were correlated to clamp-to-

clamp measurements of tendon strain (control tendons: R2=0.63, p < 0.001) the

strains in the midusbstance were typically half that of machine based tendon strains.

Some of the discrepancy between optical and machine strain measures may be at-

tributed to slippage at the tendon/clamp interface — a well known technical issue

that can plague the mechanical testing of soft tissues [21, 22]. However analysis

of video data indicated a lack of clamp slippage with our employed clamping tech-

nique [18]. Therefore we rather attribute the optical/machine strain measurement

discrepancy in the current study to relative movements between internal tendon

substructures.

From the recorded video we were able to see that murine Achilles tendons were

formed from intertwined primary fiber bundles that twisted in a coordinated fashion

under load. These bundles had unique functional axes, and were surrounded and

interconnected by fascias. Our measurements were not sufficiently sensitive to detect

fiber uncrimping [23, 24, 25] or to determine whether inter-fascicle slippage occurred

[26, 27, 28, 29]. These factors, as well as collagen fibril slippage at the ultrascale [2, 3,

30], have been described as providing a potentially important functional contribution

to macroscopic tendon behavior.

Although optical measurement of soft tissue strains is becoming more standard

and can improve analytical precision in examining tendon substructures, these mea-

40

3.1 Local strain measurement

surements are still limited by the assumption that surface strains are indicative of

the strains inside the various tendon subregions. As was alluded to above, hier-

archical collagen structures yield complex tissue stress and strain patterns due to

progressive recruitment and eventual failure of constituent fiber bundles and do not

generally represent the response of a continuum material. Despite this important

limitation we believe that useful insight into local tendon mechanics can still be

extracted.

Some biomechanical studies on the murine Achilles tendon [31, 15, 16] have re-

ported higher strains at failure, lower failure loads and a lower stiffness than was

measured in the current study. This discrepancy might be a consequence of the

experimental set-up, since our tests were performed in PBS at 37◦C, while the cited

studies reporting stiffer/stronger tendons tested at room temperature and moist-

ened with lightly sprayed PBS. Since we focus here on relative differences between

natural and GAG-depleted tendon under equivalent (and approximately physiolog-

ical) loading conditions, concerns regarding discrepancies in the mechanical testing

approach are of secondary importance.

We observed a statistically lower concentration of GAGs in the proximal subregion

compared to the two subregions closer to the bone. While this is consistent with the

doctrine that the concentration of GAGs depends on the level of compressive forces

acting in that region, the differences we observed were considerably smaller than

previously reported values of GAG content at the bone insertion [32, 33, 9]. This

discrepancy may be attributed to the fact that the tendon (midsubstance) considered

in the present study was dissected proximally from the actual bone-tendon interface

where a more fibrocartilage-like ECM is present [10].

The tensile mechanical effects of GAG digestion were indicated as a decrease in

ultimate load and elastic modulus. However, a focused analysis of strains within

three subregions of the midsubstance indicated that the change in modulus was

not uniformly distributed over the entire tendon. More precisely, the optical strains

increased toward the bone insertion upon GAG removal, while the central and proxi-

mal subregions were relatively unaffected. This inhomogeneous mechanical response

to partial GAG removal may reflect the complexity of the transition from tendon to

bone [34]. This histological and mechanical transition complicates the interpretation

of our data with respect to a mechanical (e.g. cross-linking) role of GAG in tendon.

Interpretation of the mechanical effects of enzyme digestion in the central portion

of the midsubstance is more straightforward. In contrast to the bone region the

central subregion is constituted of a largely homogenous cell phenotype, and these

fibroblast-like cells produce a parallel-fibered ECM that is optimized to effectively

41

Tissue-organ level

transmit tensile load.

Our finding that the tensile properties of the central portion of the tendon mid-

substance are insensitive to DS/CS content supports some recent mechanical studies

on individual tail tendon fascicles [15] and intact human ligaments [14], in which

GAGs have been enzymatically removed. Importantly, these findings collectively

contradict hypotheses that have emerged from modeling and ultrascale experimen-

tal studies investigating a potential mechanical role of decorin as a cross-linker that

is fundamental to tensile tendon mechanics [2, 12, 13, 11]. It is well established that

decorin structurally interconnects type-I collagen fibrils at discrete binding periods,

but our results indicate that these interconnections (or lack of them) do not quan-

titatively contribute to tensile tendon mechanics. We conclude rather that decorin

content is probably associated with other well-described functional roles such as

tissue hydration [32] and structural organization [5]. By regulating tissue water

content, cell mediation of decorin content may partially dictate tendon mechan-

ics by promoting/inhibiting inter-fibril gliding or through dissipative (viscoelastic)

mechanisms that were not directly investigated here.

In conclusion, our results indicate that GAG content may affect elastic tensile

tendon mechanics in the distal third of the midsubstance, but apparently not in

more proximal midsubstance subregions. This study presents evidence against an

essential role of PGs as collagen fibril cross-linkers in resisting tensile load. It must

be noted that more complex mechanical behaviors (such as stress relaxation, creep,

fatigue resistance, shock dissipation, etc.) may in fact be regulated by CS/DS con-

tent, and there is some evidence indicating that GAG composition reflects these

functional demands [6]. Finally, the demonstrated variability in structure and func-

tion of the various tendon subregions highlights the importance of a local strain

measurement at regions of interest and indicates that structural measurement is by

itself inappropriate for assessing tendon midsubstance behavior.

Conflict of interest statement

All authors have no conflict of interest and nothing to disclose.

Acknowledgement

This work was supported through BEST Bioengineering Cluster at ETH Zurich

(Switzerland) and the EU grant LHSB-CT-2003-503161 (Genostem). The authors

also thank Prof. Edoardo Mazza at the ETH Zurich Center for Mechanics for his

42

References

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46

Chapter 4

Material level

4.1 Collagen fibril morphology

4.1 Collagen fibril morphology and mechanical

properties of the Achilles tendon in two inbred

mouse strains

S. Rigozzi1, R. Muller1, and J.G. Snedeker1,2

1Institute for Biomechanics, ETH Zurich, Switzerland

2Orthopedic Research Laboratory, University of Zurich, Balgrist, Zurich, Switzer-

land

published in:

Journal of Anatomy

vol(216):724-731, 2010

Abstract:

The relationship between collagen fibril morphology and the functional behavior of

tendon tissue has been investigated in numerous experimental studies. Several sug-

gest that larger fibril radius is a primary determinant of higher tendon stiffness and

strength; others have shown that factors apart from fibril radius (such as fibril-fibril

interactions) may be critical to improved tendon strength. In the present study, we

investigate these factors in two inbred mouse strains that are widely used in skeletal

structure-function research — C57BL/6J (B6) and C3H/HeJ (C3H). The aim was

to establish a quantitative baseline that will allow one to assess how regulation

of tendon extracellular matrix architecture affects tensile mechanical properties.

We specifically focused on collagen fibril structure and glycosaminoglycan (GAG)

content — the two primary constituents of tendon by dry weight — and their

potential functional interactions. For this purpose, Achilles tendons from both

groups were tested to failure in tension. Tendon collagen morphology was analyzed

from transmission electron microscopy (TEM) images of tendon sections perpen-

dicular to the longitudinal axis. Our results showed that the two inbred strains are

macroscopically similar, but with higher elastic modulus in C3H mice (p < 0.05).

Structurally, C3H mice showed a larger collagen fibril radius compared to the B6 (96

± 7 nm and 80 ± 10 nm respectively). Tendons from C3H mice also showed smaller

specific fibril surface (0.015 ± 0.001 nm/nm2 vs. 0.017 ± 0.003 nm/nm2 in the

49

Material level

B6 tendons, p < 0.05), and accordingly lower concentration of GAGs (0.60 ± 0.07

µg/mg vs. 0.83 ± 0.11 µg/mg, p < 0.05). As in other studies of tendon structure

and function, larger collagen fibril radius appears to be associated with stiffer

tendon, but this functional difference could also be attributed to reduced potential

surface area exchange between fibrils and the surrounding proteoglycan rich matrix,

in which the hydrophilic GAG side chains may promote inter-fibril sliding. This

study provides an architectural and functional baseline for a comparative murine

model that can be used to investigate the genetic regulation of tendon biomechanics.

Keywords:

Achilles tendon, biomechanics, collagen fibrils morphology, glycosaminoglycans, in-

bred strain

Introduction

The relationship between the distribution of collagen fibrils and functional behavior

of tendon has been investigated in numerous experimental studies. Several of these

suggest that increased fibril radius is a primary determinant of improved tendon stiff-

ness and strength [1, 2, 3, 4]. More recent studies have shown however, that despite

a generally smaller fibril radius in healing tendon, it gains strength with an increase

in the fibril area fraction, and that factors apart from fibril radius may be critical

to improved tendon strength [3]. Consistent with this concept, an earlier study in-

dicated that Achilles tendons of growth differentiation factor 5 (GDF-5) deficient

mice showed drastically reduced functional performance while being composed of

nearly normal fibril radius distributions and fibril area fractions [5]. Generally these

studies indicate that biochemical deficiencies in the non-collagenous extracellular

matrix (ECM) may be a primary causative factor in certain tendon pathologies.

Two main classes of extracellular macromolecules make up the tendon matrix:

proteoglycans (PGs), which play a complex role in force transmission and mainte-

nance of tendon tissue structure [6, 7], and collagen fibrils. Studies that have investi-

gated the relationship between structural and mechanical properties have generally

focused on one major component; either collagen or PGs, with studies focusing on

collagen fibril morphology being more common. Some of the earliest studies have

related collagen fibril morphology and mechanical properties [1, 8, 9], and these

suggest that tendon ability to bear high stress is positively related to the percent-

age of large fibrils in the tissue, while creep-inhibition properties are related to the

percentage of small fibrils. These studies suggest that tendon is thus able to fulfill

50

4.1 Collagen fibril morphology

its specific functional demands by regulating an appropriate collagen architecture.

On the other hand, various studies report that a high concentration of negatively

charged PG side chains — the glycosaminoglycans (GAGs) — is related to an in-

creased osmotic pressure, and enhanced tissue hydration [10]. This tissue hydration

could separate individual fibril bundles and minimize shear stress as fibrils move

relative to one another [11]. In other words, PGs may lubricate the structure at

the molecular level, facilitate inter-fibril slippage, and thus make the tissue more

compliant.

In the present study, we evaluated the ultrastructural and mechanical properties

of the Achilles tendon in the two inbred mouse strains C57BL/6J (B6) and C3H/HeJ

(C3H). Adult B6 and C3H mice have a similar body size and weight and their skele-

tons are similarly sized, but they show morphologically distinct skeletal traits [12].

Despite differences in adult peak bone density and whole bone cross-sectional area,

they have similar bone mechanical properties at the structural level [13]. We hy-

pothesized that, as with bone, these two genotypes would show differences in tendon

architecture while exhibiting similar macroscopic mechanical behavior — and thus

provide a useful comparative (genetic) model for investigating tendon structure-

function. In quantifying tendon structure, we focused on defining the collagen fibril

morphology in terms of both fibril radius and the potential for fibril-to-fibril in-

teractions. This allows one to extract information about the relative functional

contribution of not only the collagen, but also other macromolecules like the PG

decorin, which attaches to the surface of the collagen fibrils at regular intervals [14].

The aim of this study was to thus define an architectural and functional baseline

for comparison between two inbred mouse strains as a model for a genetic basis of

tendon structure and function.

Materials and Methods

Animal model

This study was reviewed and approved by the local and state authorities for all levels

of animal investigation. We used two inbred mouse strains with markedly different

skeletal phenotypes, where B6 represents the low bone mass and C3H represents the

high bone mass phenotype [12, 15, 16, 17, 18]. Ten B6 and ten C3H female mice were

purchased from the Harlan breeding facility (Horst, The Netherlands). Mice were

sacrificed by CO2 inhalation at 19 weeks of age. The animals were then stored at

-20◦C and thawed at room temperature just before dissection of the Achilles tendon.

Twenty Achilles tendons were harvested from each of the two strains.

51

Material level

Collagen fibril structure analysis

The tendon connected to the soleus muscle was dissected and fixed in a solution

of 2% glutaraldehyde for 30 minutes and rinsed three times with 0.1M cacodylate

buffer (pH 7.4). After fixation, tendons were postfixed with 2% osmium oxide for

30 minutes and then rinsed three times in distilled water. The samples were stained

with ethanolic uranyl acetate (2% uranyl acetate/50% ethanol) overnight and then

twice rinsed with distilled water. Dehydration was continued through a cold (4◦C),

graded ethanol series. The tendons were infiltrated and embedded through propylene

oxide in a fresh mixture of Epon and ethanol; polymerized for 2 days in the oven at

60◦C.

Figure 4.1: Representative transmission electron micrograph for the two inbredstrain; B6 and C3H, and its binarized reconstruction on the right. Thebinarized reconstruction is processed to calculate the fibril radius, thecollagen area fraction, the interfibrillar distance, the specific fibril surfaceand the fibril contact area. The color bar indicates the pixel size of thefibril radius that is converted to nm. The black bar indicates 400 nm.

52

4.1 Collagen fibril morphology

Figure 4.2: Schematic representation of the morphological parameters measuredwith the automatic script. (A) Fibril radius, (B) collagen area fraction,(C) interfibrillar distance, (D) specific fibril surface, (E) fibril contactarea.

Sections were cut with 50 to 70 nm thickness perpendicular to the longitudinal

axis of the tendon with a diamond knife mounted to an ultramicrotome (Reichert

Ultracut E, Wetzlar, Germany) Sections taken approximately 1 mm proximal from

the calcaneous attachment were mounted to Butvar-coated 1 × 2 mm slot grids,

contrasted with 2% uranyl acetate and lead citrate according to the method of

Reynolds [19]. The region of interest (ROI) was identified at ×5’000 magnification.

Using a transmission electron microscope (TEM), seven micrographs per tendon

were then taken at a final magnification of ×40’000 as shown in Figure 4.1. The

fourteen animals thus yielded a total of 98 micrographs at 100 kV.

A script was written in Matlab (The MathWorks, version 7.4) to automatically

quantify morphological parameters, as schematized in Figure 4.2. The original image

was automatically calibrated, treated with a Gauss filter (width, σ = 1.2; support,

s = 3 voxels), and thresholded according to the algorithm of Trussell [20] to give

a binary image that was stored and used to measure the geometric parameters of

the fibrils. The measured parameters described below can be divided in two groups:

classic parameters widely described for collagen morphology characterization (col-

lagen fibril radius, collagen area fraction), and newly introduced morphometric pa-

rameters describing the potential for lateral interactions between the collagen fibrils

(interfibrillar distance, specific fibril surface, fibril contact area). The collagen fib-

ril radius of each collagen fibril was represented by the minor collagen radius, to

avoid any error due to oblique fibril angle in relation to the image plane. Addition-

ally, the fibril radii of the 7 images from each animal were cumulatively summed to

give an overall indication of the distribution. The cumulative curves were shown to

give a supplementary overall indication of the distribution within the two strains.

Collagen area fraction was defined as the total fibril area in the image divided

by the total image area (yielding a dimensionless parameter). The interfibrillar

53

Material level

distance was defined as the mean spacing between fibrils expressed in nm. This

value was calculated by applying a distance transform on the gap between the fib-

rils [21], resulting in the average distance between a given fibril and all neighboring

fibrils that can be connected by an uninterrupted line segment (Figure 4.2C). The

specific fibril surface, expressed in nm/nm2, is a normalized value consisting of

the total perimeter of all fibrils within a given image divided by the total image area

(Figure 4.2D). The fibril contact area is defined as the percentage of the total

fibril perimeter that is in contact with neighboring fibrils (Figure 4.2E). Two fibrils

are considered in contact if the total perimeter of each cannot be distinguished as an

isolated circle on a binary image. The exchange area between the collagen fibrils and

the outer ECM is calculated by applying a Canny filter on the filtered image [22],

the Canny filter detects the edges of the fibrils by applying two thresholds to the

gradient: a high threshold for low edge sensitivity and a low threshold for high edge

sensitivity. The filter starts from the low sensitivity and then grows it to include

connected edge pixels from the high sensitivity result to fill in gaps in the detected

edges.

Table 4.1: Summary of measured morphological parameters measured for B6 andC3H mice

Mean radius

(nm)

Collagen area

fraction (%)

Interfibrillar

distance (nm)

Specific fibril sur-

face (nm/nm2)

Fibril contact

area (%)

C57BL/6 79.7±9.7* 72.5±3.8* 10.8±1.6 0.017±0.003* 33.0±4.2

C3H/HeJ 95.7±7.0* 73.1±3.0* 10.9±2.1 0.015±0.001* 34.5±2.5

* Statistical significant for p < 0.05

GAG analysis

To relate the morphological measures of potential for fibril-to-fibril interactions to

PG content, we assessed GAG content. Tendons were weighed and digested for 16 h

with 500 U/ml papain solution in buffer (0.1M disodium hydrogen phosphate, 0.01M

EDTA disodium salt, 14.4mM L-cysteine) at 60◦C. GAG content was determined

spectrophotometrically (Cary 50, Varian, Zug, Switzerland) at 525 nm following

binding to dimethylmethylene blue dye (DMMB) [23] using chondroitin sulfate as

the standard.

Cupromeronic blue dye (CB) was used to visualize GAG architecture with TEM

according to reported studies [24, 25]. Briefly, freshly dissected Achilles tendons

connected to the soleus muscle were stained overnight with 1% CB in 0.2M acetate

54

4.1 Collagen fibril morphology

buffer (pH 5.6). After rinsing in the same solution without CB, samples were im-

mersed in 0.5% Na2WO4 in buffer for 1 h and then overnight in 0.5% Na2WO4 in

30% ethanol. To stain the collagen fibrils, samples were stained with 1% uranyl

acetate and fixed with the Reynolds technique as mentioned above [19].

Biomechanical testing

Mechanical tests were performed with a universal testing machine (Zwick 1456,

Ulm, Germany). Tendons were preconditioned with 10 cycles of 0-10% nominal

strain before ramp loading to failure at 10 mm/min, as described earlier [7]. Force

displacement data from the ramp-to-failure test were analyzed for ultimate stress,

failure strain, and elastic (E) modulus.

Statistical analysis

A Lilliefors test was used to determine whether data were adequately fitted to a

normal distribution. For normally distributed data, an unpaired t-test was used to

test the significance of the two strains on measured morphological and mechanical

parameters. Otherwise a Wilcoxon rank sum test was used to test significance

between the strains. A p-value < 0.05 was considered to be significant.

Two-sample Kolmogorov-Smirnov testing was used to compare the distribution

of the collagen fibril mean radii between the cumulative curves of the two inbred

mouse strain. The distributions were considered significantly different for p-values

< 0.05.

Results

Collagen fibril structure analysis

Images from the two inbred strains were compared, and the results are summarized

in Table 4.1. Results show that C3H mice have a significantly larger mean fibril

radius than B6, while the collagen area fraction of the ECM and the lateral spacing

between fibrils can be considered the same for both groups. Specific fibril surface was

significantly higher in the B6 strain, but effective fibril contact area was statistically

similar. Figure 4.3 shows cumulative distribution curves for fibril radii for each

mouse. The curves for the B6 strain have a steeper slope, indicating that they

contain more small-radius fibrils than the C3H. There was a statistically significant

inter-strain difference for the curves.

55

Material level

Figure 4.3: Cumulative distribution curves for B6 and C3H mice. B6 generally con-tain a majority of small fibrils, while C3H have a more uniformly dis-tributed fibril radius. Histograms of the collagen fibril distribution forboth groups. n is the total number of fibrils.

GAG analysis

The GAG assay indicated a statistically significant difference in GAG concentration

(chondroitin sulfate and dermatan sulfate); for B6 0.83 ± 0.11 µg/mg and for C3H

0.60 ± 0.07 µg/mg (p < 0.05). Representative transmission electron micrographs of

the longitudinal sections of the two inbred strain are shown in Figure 4.4.

Biomechanical testing

During mechanical testing, all tendons failed in the midsubstance. Video analysis

revealed no slippage at the clamp. The mechanical parameters are summarized in

Table 4.2. Both groups had a similar tendon length (5.5 ± 0.7 mm for the B6 and

5.5 ± 0.6 mm for the C3H), and cross-sectional area (0.49 ± 0.10 mm2 for the B6

and 0.54 ± 0.14 mm2 for the C3H). The C3H group exhibited a median E-modulus

of 140 MPa while B6 was significantly lower; 123 MPa (p < 0.05). No statistically

significant difference in either mean failure strain (εB6 = 36 ± 17 % εC3H = 33 ± 9%)

or mean ultimate stress (σB6 = 10 ± 4 MPa σC3H = 13 ± 4 MPa), was observed.

56

4.1 Collagen fibril morphology

Figure 4.4: Representative transmission electron micrograph of a longitudinal sec-tion of the Achilles tendon for (A) B6 and (B) C3H at a magnificationof ×53000. The black lines between the fibrils are the stained GAGs.(C) Means and standard deviations of sulfated GAG content in the twogroups. * statistically significant for p-value < 0.05.

Assessing structure and function

Table 4.1 and Table 4.2 summarize all the measured values of the two strains of

inbred mice. The two groups had similar gross anatomical characteristics, with

no statistical difference seen in cross-sectional area, or tendon length at the given

pre-load. No differences were observed in stiffness, maximal force and max strain.

Mechanically, the C3H showed a statistically higher elastic modulus than B6 (p <

0.05). Ultimate stresses were higher in the C3H, but this was not statistically

significant.

Data demonstrated morphological differences at the collagen fibril level. The C3H

group had significantly larger mean fibril radii and lower fibril perimeter than the B6,

while the B6 showed a higher specific fibril surface (p < 0.05). Other morphological

parameters such as collagen area fraction, interfibrillar distance and fibril contact

area did not show statistical differences.

Thus, the B6 group tendons were similarly sized, but generally less stiff than

the C3H tendons, which had fewer, larger collagen fibrils and smaller potential for

lateral interactions between fibrils or between fibrils and the other components of

the ECM.

57

Material level

Table 4.2: Summary of the measured mechanical parameters for B6 and C3H mice.The E-modulus is showed as a median (first quartile; third quartile).

Structural data

Groups L0 (mm) Area (mm2) Force max

(N)

Failure strain

(%)

Stiffness

(N/mm)

C57BL/6 5.5±0.7 0.54±0.14 6.0±2.3 36±17 52±36

C3H/HeJ 5.5±0.6 0.49±0.10 6.4±1.8 33±9 75±28

Material data

Groups Ultimate

stress (MPa)

E-modulus

(MPa)

C57BL/6 10.4±3.9 123 (52;203)*

C3H/HeJ 13.0±3.7 140 (112;198)*

* Statistical significant for p < 0.05

Discussion

Tendon stiffness is central to its proper function [26] and how tendon structure relates

to function is critical to understanding tendon pathology [27, 28, 29, 30] as well as

in developing strategies for healing [3, 31]. In this work, we introduce a potentially

useful comparative tendon structure-function model in two inbred mouse strains by

describing the collagen morphology and the corresponding mechanical properties of

the soleus tendon. The mechanical properties of this load bearing, elastic tendon

were considered with respect to the observed morphology of the collagen fibrils,

where it was found that tendons with larger diameter collagen fibrils and lower

specific fibril surface also exhibited a higher elastic modulus.

Previous studies have investigated the morphology of collagen fibrils from cross-

sectional area TEM images. Some have focused on the collagen radius in ruptured

tendons [32, 27], some on the changes to the collagen during collagen assembly

[33]. Others have focused on the collagen fibril diameters in different anatomical

structures and their relationship to corresponding function [34]. In all of these

studies fibril morphology has been described primarily in terms of fibril diameter.

In the current study we introduce new morphological parameters that permit insight

into potential fibril-fibril interactions and fibril-non-fibrillar matrix interactions such

as the interfibrillar distance between the fibrils, the specific fibril surface, and direct

fibril-fibril contact (see Figure 4.2, and Table 4.1). These parameters not only give

a more thorough description of the collagen morphology, but may also offer better

insight into structure-function relationships.

Studies analyzing structure-function in tendons usually separately consider the

58

4.1 Collagen fibril morphology

functional relevance of collagen fibril morphology and proteoglycan content, and thus

neglect potential functional interplay between collagen and PGs. By expanding our

quantification of collagen fibril morphology to include descriptors relevant to fibril-

fibril interactions, we have attempted here to better bridge conceptual treatment of

these two components that are known to influence each other during tissue formation

[10, 35, 36], and may influence each other with regard to mechanical function [37,

38, 39, 7, 40].

Our mechanical and morphological comparison between B6 and C3H showed that

despite very similar collagen area fractions, C3H tendons are considerably stiffer

than the B6. On one hand, this could be attributed to larger fibril size; the B6

mouse strain has Achilles tendons constituted by smaller fibrils. Alternatively, the

lower modulus in the B6 mice may also be partly explained by greater collagen fibril

surface area and fibril-hydrated ECM interactions; since collagen fibrils are densely

linked to hydrophilic PG chains where their surface is in contact with the matrix,

these chains may facilitate relative movement of the fibrils through the matrix [41].

Considering that the collagen fibrils are linked to PG chains where their surface

is in contact with the matrix, we surmise that the amount of PGs present in the

tendon is directly correlated to the relative fibril surface area. Our results described

in Table 4.1 and Table 4.2 show that C3H mice, which have larger E modulus

and lower total fibril surface area, also have a lower measured concentration of

sulfated groups (DS and CS). Thus, less PG may be associated with a higher elastic

modulus. As discussed below, this is in contrast with other studies that invoke the

structure function model that the PGs may actually interconnect neighboring fibrils

[42, 43, 14, 44], transfer forces between them [38, 45], and lead to stiffer tendons

[39].

Given comparable macroscopic/microscopic tendon architecture in the two evalu-

ated inbred strains, classic composite theory suggests that the observed similarity in

collagen area fraction (e.g. effective collagen content) in both groups would yield a

similar mechanical response in terms of elastic modulus and failure stress [46, 47, 41].

In fact, the C3H demonstrated significantly higher elastic modulus, but a similar

failure behavior. This suggests that a more sophisticated model of tendon structure

function is required.

More elaborate models of tendon structure-function have been introduced, taking

into account fibril-fibril interactions through matrix cross-linking by proteoglycan

GAG sidechains [48, 39]. In the present study, the close similarity in both mean

interfibrillar distance and fibril-fibril contact area suggests that there would be no

relative advantage in either group with regard to direct fibril-fibril interactions (lat-

59

Material level

eral force transfer between fibrils) that might promote a higher elastic modulus of

the C3H tendons. In fact according to this model the higher relative GAG content

in the B6 mice should confer stiffer tendon properties, which is in direct contrast

to our experimental observations. Thus the present study suggests that tendon

structure-function is also not adequately described by the theoretical framework of

proteoglycan mediated collagen fibril load sharing. This lends support to our recent

studies indicating that GAG side chains of small leucine rich proteoglycans do not

play a dominant role in tensile mechanics of the tendon midsubstance [7, 49].

Until now, most studies investigating GAG mediated fibril-fibril interactions have

relied upon either PG knock-out mice [40] or enzyme digestion of the PG side chains

[50, 51, 7, 49]. Cross-sectional comparisons of structure and function in two differ-

ent inbred wild type strains can complement such studies by avoiding problematic

limitations inherent in these approaches. For instance, a “targeted” enzymatic di-

gestion of the GAG component does not guarantee that another component will not

be affected and show secondary effects, such as swelling of the collagen fibers [52].

Also in studies with knock-out mice, the lack of a certain gene does not ensure that

redundant processes will not compensate its loss and restore function [53, 40].

Our choice of inbred mouse strains with regard to a structure-function baseline

in tendon is rooted within a history of studies investigating bone morphology of

different inbred strains to correlate them with their mechanical response [12, 15, 16,

17, 18]. B6 and C3H have been identified as a model to study the genetic factors in

osteoporosis [54]. Since tendon to bone healing remains a pressing clinical challenge

[55, 56], this model may be enlarged to permit a broader view of how these two

connective tissues influence each other.

It should be noted that our previous studies have revealed that heterogeneous

histological structure (e.g. tendon regions nearer to the bone and muscle insertions)

has important implications with regard to the mechanical influence of proteoglycans

on tensile mechanics [7]. The aim of the present work was to focus on a specific

subregion of the tendon with more homogeneous tissue architecture, in hopes of

reducing the parameters considered in our structure-function analysis. While we

intentionally focused the scope of the present investigation to the tendon midsub-

stance, quantitative investigation of the transition between tendon and muscle or

tendon and bone remains ground for future work.

To conclude, inbred C3H mice have higher elastic modulus, larger mean fibril ra-

dius, lower fibril surface area, lower GAG content, similar collagen area fractions,

and similar direct fibril-fibril contact compared to B6 mice. This study thus estab-

lishes a useful baseline for further investigation of the influence of tendon morphology

60

References

on tendon mechanical properties in two inbred strain populations that are increas-

ingly used as models for orthopedic disease. This baseline forms a foundation for

future studies into the genetic regulation of tendon modeling and remodeling. It

will also aid in the interpretation of existing studies that have employed selective

chemical degradation of tendon ECM components, and transgenic animal models

used to understand how tendon derives its mechanical characteristics from its basic

architecture and biochemical composition.

Acknowledgements

This work was supported through BEST Bioengineering Cluster at ETH Zurich

(Switzerland) and the EU grant LHSB-CT-2003-503161 (Genostem).

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66

4.2 Mechanical response of individual collagen fibrils

in loaded tendon as measured by atomic force

microscopy

S. Rigozzi1, A. Stemmer2, R. Muller1, and J.G. Snedeker1,3

1Institute for Biomechanics, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich,

Switzerland

2Nanotechnology Group, ETH Zurich, Tannenstrasse 3, 8092 Zurich, Switzerland

3Orthopedic Research Laboratory, University of Zurich, Balgrist, Forchstrasse 340,

8008 Zurich, Switzerland

published in:

Journal of Structural Biology

vol(176):9-15, 2011

Abstract:

A precise analysis of the mechanical response of collagen fibrils in tendon tissue

is critical to understanding the ultrastructural mechanisms that underlie collagen

fibril interactions (load transfer), and ultimately tendon structure-function. This

study reports a novel experimental approach combining macroscopic mechanical

loading of tendon with a morphometric ultrascale assessment of longitudinal and

cross-sectional collagen fibril deformations. An atomic force microscope was used

to characterize diameters and periodic banding (d-period) of individual type-I

collagen fibrils within murine Achilles tendons that were loaded to 0%, 5%, or 10%

macroscopic nominal strain, respectively. D-period banding of the collagen fibrils

increased with increasing tendon strain (2.1% increase at 10% applied tendon strain,

p < 0.05), while fibril diameter decreased (8% reduction, p < 0.05). No statistically

significant differences between 0% and 5% applied strain were observed, indicating

that the onset of fibril (d-period) straining lagged macroscopically applied tendon

strains by at least 5%. This confirms previous reports of delayed onset of collagen

fibril stretching and the role of collagen fibril kinematics in supporting physiological

tendon loads. Fibril strains within the tissue were relatively tightly distributed

Material level

in unloaded and highly strained tendons, but were more broadly distributed at

5% applied strain, indicating progressive recruitment of collagen fibrils. Using

these techniques we also confirmed that collagen fibrils thin appreciably at higher

levels of macroscopic tendon strain. Finally, in contrast to prevalent tendon

structure-function concepts data revealed that loading of the collagen network is

fairly homogenous, with no apparent predisposition for loading of collagen fibrils

according to their diameter.

Keywords:

collagen fibril, atomic force microscope, collagen network, tendon

Introduction

The mechanical properties of tendon must be optimized to avoid injury and effi-

ciently transfer load from muscle to bone [1]. Tendon is hierarchically structured

(Figure 4.5), being mainly composed of collagen fibrils that are organized into func-

tional units (fibers, or fascicles) which are further organized according to the specific

functional demands of the tendon itself [2]. As the primary load-bearing protein in

tendon, the nature of collagen load distribution has been widely investigated at

multiple size scales [3, 4, 5, 6]. Particular attention has been directed toward un-

derstanding the behavior of the collagen fibrils themselves [7] as well as how the

bundled collagen fibrils collectively respond to applied macroscopic loads [8].

At the macroscopic scale, the most common structure-function analyses of tendon

have been performed using tensile test to failure of single tendon fibers (fascicles)

extracted from rat tail tendon [9, 10]. More complex anatomical structures composed

of intertwined fibers have also been characterized [11, 12] and modeled [13, 14]

in efforts to understand the interaction between these structures under load. It

has been reported that only 40% of macroscopic (fiber level) tendon elongation

can be attributed to straining of individual collagen fibrils, and the majority of

tendon strain would thus appear to be due to inter-fibril movements, such as the

progressive alignment of the collagen fibrils in the direction of applied load [8, 3,

7]. Although mechanical properties of single fibrils have been investigated by one-

dimensional tensile testing using optical tweezers [15] and atomic force microscopy

(AFM) [16, 17], the nature by which collagen fibrils interact within their network

to accommodate applied tendon strains remains largely unknown [3]. Part of this

uncertainty lay in the limited ability of current methods to discern in situ strains in

individual collagen fibrils.

68

4.2 Collagen fibrils’ role in tendons behavior

Macroscopic tendon extension is enabled by straining and sliding mechanisms that

simultaneously occur at different length scales. At the tissue level this is enabled by

straining of and sliding between collagen fibers (Figure 4.5). Within the fibers, ultra-

scale straining and sliding of collagen fibrils also occurs, and this is partly regulated

by the proteoglycan rich, non-collagenous matrix [18, 11, 19]. The relative straining

associated with collagen molecule stretch can be monitored by assessing d-period

length changes [3, 7].

Attempts to directly relate tendon ultrascale structure to tissue-scale function

have employed various experimental approaches including optical and scanning elec-

tron microscopy of sectioned tendon tissue [20, 21, 22], AFM on isolated fibrils

[16, 23], or scattering X-ray spectroscopy on tendon fibers [8, 3, 24, 7]. Scanning

electron microscopy studies of tendon sections allow excellent visualization of the

collagen structures, but quantitative measures of collagen fibril dimensions (and

eventually mechanical collagen strains) is limited by perspective distortion. Trans-

mission electron microscopy (TEM) studies are better suited for morphometric anal-

ysis, but due to difficulties in aligning the sectional plane with the longitudinal axis

of collagen fibrils, have been used almost exclusively for morphometric analysis of

collagen fibril cross-section [25, 26]. X-ray spectroscopy has been successfully used

to infer collagen fibril strain in loaded tendon fibers [8, 3, 7], but visualization of

individual fibrils is not possible, precluding direct investigation of collagen network

deformation. Finally, while AFM can provide very precise morphometric quantifica-

tion of single fibril collagen topology and mechanics [16, 23], AFM has not yet been

applied in studying collagen fibril strains in loaded tendon. Thus no study has yet

implemented a method to accurately quantify the ultrascale mechanical response of

individual collagen fibrils embedded within a loaded tendon.

The motivation for this study was therefore to introduce a metrically accurate

AFM visualization approach to characterize mechanical strain response of individual

collagen fibrils within the fibril network comprised by a tendon fiber. We specifically

hypothesized that such an approach might elucidate the reported lag between col-

lagen fibril strain and applied tendon load by providing a more direct visualization

of fibril load distribution within the network. To investigate this hypothesis, we

measured collagen fibril strains in populations of individual fibrils within a loaded

tendon, relying on d-period banding distance as a proxy measure of collagen fibril

strain.

69

Material level

Materials and Methods

Sample preparation

All animal experiments were reviewed and approved by local and state authorities

(Kantonales Veterinaramt Zurich, Zurich, Switzerland). Achilles tendons were har-

vested from 19-week-old female C57BL/6 inbred (wild type) mice. These inbred

mice were selected as an experimental model for their strong genomic homology to

humans [27, 28, 29], for their low genetic variability, and for their potential use as

a genetic background in future structure-function investigations using knock-out or

transgenic mice. Each tendon was laid onto a piece of paper and mounted in a saline

filled testing chamber designed to prevent sample dehydration. Mechanical tests

were performed with a universal testing machine (Zwick 1456, Ulm, Germany). Us-

ing previously described methods [26], five (n=5) tendons were preconditioned with

10 cycles of 0-10% nominal strain (defined as incremental tendon elongation under

load, normalized as a percentage of the original unloaded tendon length) before

ramp loading to failure. From these curves the “linear” region of the tendon mate-

rial response (Figure 4.5) corresponding to reversible (non damaging) deformation

at the fibril level [8] was defined for subsequent investigations. Later tendon test

groups (n=5 each) were stretched to target strain levels in 5% increments covering

this range (i.e. 0%, 5%, and 10% nominal strain). All tendons were fully hydrated

in phosphate buffered saline (PBS) solution prior to chemical fixation with 1.25%

glutaraldehyde solution (G5882, Sigma, St. Louis, MO) added to the test chamber

just prior to application of mechanical loading to the target strain. Tendons were

then maintained under constant load for 8 hours until the structure was fixed [30].

The applied load and corresponding mechanical strain over eight hours were

recorded for each tendon (see Figure 4.5). Afterwards the tissue was rinsed with

PBS, embedded in Tissue-Tek (Tissue-Tek, 4583 Compound, Sakura Finetek Eu-

rope, Zoeterwoude, Netherlands) for at least 12 h at -20◦C and sectioned in the

longitudinal plane with a cryostatic microtome (SLEE, Mainz, Germany) at -20◦C

[31]. Twenty micrometer thick frozen sections were attached to a polylysine-coated

glass slide (SuperFrost, Menzel-Glaser, Braunschweig, Germany) and dehydrated

in an oven at 60◦C. For each fixed tendon, 3 slices from the tendon midsubstance

(central third) over a length of 60 µm were analyzed as described below.

70

4.2 Collagen fibrils’ role in tendons behavior

Figure 4.5: Experimental setup for fixing the strained tendon at a given load. Thetendon is clamped to a universal tensile test machine in a chamber thatis filled with 1.25% glutaraldehyde solution, while the tendon is underconstant load for 8 h. The applied constant load (input) and the tendonstrain response (output) are recorded. The fixed tendon is sliced andthe fibril strain is measured with an AFM. The images show the d-period observed with AFM, scanning electron microscopy (SEM), andtransmission electron microscopy (TEM).

Atomic force microscopy measurements

Possible differences in the d-period between different parts of the same tendon were

measured by comparing planar sections cut parallel to the load axis. AFM exper-

iments were carried out with an Asylum Research MFP-3D-SA (USA), at room

temperature, using titanium and platinum coated silicon AFM tips on a triangular

cantilever with a nominal spring constant of 2.35 N/m (NSC11/Ti-Pt; MikroMasch,

Madrid, Spain). Images of collagen fibril topography were acquired in contact mode

at a scanning rate of 0.5 Hz in air on the dehydrated thin-sectioned samples.

From each slice, a minimum of five 2µm × 2µm regions of interest (ROI) were

analyzed. All height images were flattened and equalized with WSxM 4.0 Develop

11.4 software (Nanotec Electronica S.L.) [32], as displayed in Figure 4.6a. For each

ROI, between 5 and 10 fibrils were randomly identified for analysis. For each ana-

lyzed fibril, a line of analysis was selected on the long-axis of the fibril in the X-Y

71

Material level

Figure 4.6: (A) AFM height image of collagen fibrils taken in contact mode, (B)two-dimensional profile of the z-axis corresponding to the line on imageA. (C) Detail of a typical power spectrum density of the 2-D profile; thehighest peak in the graph corresponds to most frequent periodicity inthe two-dimensional profile of B. The standard dimension of the x-axisis 1/nm, in this case the inverted scale is also displayed below, and thepeak corresponds to 66.4 nm.

imaging plane, and the two-dimensional profile in the z-axis was plotted (see Figure

4.6b). The d-period was then calculated from this plot using a Fourier transform to

reveal the periodic banding frequency [23], such as that shown in Figure 4.6c. Re-

producibility tests were performed by measuring the same fibril seven times starting

from the capture of two-dimensional profile to the d-period measurement. The mea-

surement of the d-period length for a single fibril was found to be reproducible within

0.3%. Intra-image (or intra-ROI) differences in the d-period length were measured

to investigate differential responses among neighboring collagen fibrils. If the mea-

sured strains could not be fit with a normal distribution, they were considered as an

inhomogeneous response to the applied load. Intra-tendon differences in mechanical

response were also statistically tested to analyze strain differences in the anatomical

regions (i.e. regions randomly distributed on the whole tendon area).

To compare across tendon strain conditions (0% 5% 10% strain groups), all d-

period lengths of measured collagen fibrils within a given tendon were pooled. The

reference d-period length in tendons fixed at 0N was determined and afterwards used

as the control group for statistical comparison. All d-period lengths were accordingly

normalized to the control d-period length to calculate the d-period strain.

Fibril diameter and spacing between neighboring fibrils was also measured in both

unloaded controls and loaded test samples. Here the diameters of the fibrils were

measured using the cross-sectional profile of the fibril as shown in Figure 4.7 [23, 33].

72

4.2 Collagen fibrils’ role in tendons behavior

Only fibrils with clearly exposed surface contours were considered in the diametric

analysis. The diameter was measured as the transverse distance (perpendicular to

the long fibril axis) between the troughs in the tip-trace corresponding to the sides

of a fibril profile.

Figure 4.7: (A) AFM height image of collagen fibrils taken in contact mode, (B) thetwo-dimensional profile of the dash line, and (C) schematic interpreta-tion of fibril arrangement from the profile. Some fibrils can be easilyrecognized on the profile (e,f,g,h), while others have a more complicatedarrangement and do not show a clear round profile. In (C) the 2D profilefrom inset (B) is displayed to show the correspondence with the collagenfibril diameter. On the fibrils the AFM trace is drawn to scale, whilein the line above the trace is amplified 33 times. The pink semi-circlesrepresent the tip in contact to the surface.

Statistical analysis

The d-period measurements collected for each ROI were analyzed with a Lilliefors

test to examine homogeneity of fibril loading within the ROI. Homogeneity across

different anatomical regions of a given tendon was similarly investigated using Lil-

liefors test to verify data normality. Finally, Lilliefors test was performed to verify

73

Material level

Table 4.3: Relative homogeneity of collagen fibril loading in tendon (indicated by% of normally distributed measures). While some heterogeneity was ob-served in isolated regions of analysis in loaded tendons, collagen fibrilsstrains were generally homogeneous.

Tendon strain Tendon (normally distributed) ROI (normally distributed)

0% 100% 100%

5% 100% 83.3%

10% 100% 87.5%

that data distributions within test groups (0%, 5%, 10% applied strain) appeared

normally distributed. In this case, a pairwise t-test with Bonferroni adjustment

was then used to examine between-strain-group differences in fibril strains and fibril

diameters. A p-value < 0.05 was considered to be significant.

Results

Load-strain curves during tendon loading to a target strain were typical of native

(non-fixed) tendons, indicating a physiological material response during initial load-

ing to the target strain. Load-time and strain-time curves (Figure 4.5), indicated

that additional strain of the tendon under constant load (i.e. creep) was largely

arrested by glutaraldehyde fixation within the first 20 minutes, and reached a near

plateau after approximately 5 hours. AFM analysis of collagen fibril strains in a

given region of interest (randomly sampled 2µm x 2µm ROI, yielding measures

of 5 to 10 individual fibrils) revealed that fibril strains appeared to be consistently

(and normally) distributed both within individual ROI and across the various ROI’s

from an individual tendon (Table 4.3). This indicates that the collagen fibril loading

within a tendon was fairly uniform, and did not vary according to anatomical lo-

cation. Further, fibril mechanical conditioning within the experimental groups was

apparently uniform, with little variability in the d-period fibril distribution seen in

tendons loaded to the same target strain (Figure 4.8).

In mechanically strained tendons, d-period length was observed to increase with

increasing applied strain (Figure 4.8, Table 4.4). The mean d-period length of

unloaded tendon was l0% = 64.45 ± 1.83 nm. Collagen fibrils trended toward to an

elongation of 0.9% (l5% = 65.01 ± 1.68 nm, p=0.15) at an applied tendon strain

of 5% — corresponding to the end of the toe region of the tendon material curve.

Fibril elongation was only observed to statistically differ for applied strains of 10%

74

4.2 Collagen fibrils’ role in tendons behavior

Table 4.4: Summary of fibril structure characterization. A,B,C,D statistical significantfor p-value < 0.05

Target tendon strain 0% 5% 10%

D-period length (nm) 64.45±1.83A 65.01±1.68B 65.89±1.09A,B

Intrafibril elongation 0.0% 0.9% 2.1%

Fibril diameter (nm) 193.63±35.52C 195.58±32.25D 175.34±27.68C,D

(l10% = 65.89 ± 1.09 nm, p < 0.05), corresponding to the linear region of the tendon

material curve. Progressive collagen fibril recruitment was indicated both by a shift

toward larger fibril strains and a narrowing of the fibril strain distribution at higher

tendon strain (10% applied strain).

In unloaded tendons, collagen fibrils generally appeared to lie parallel to each other

and the D-periods were observed as wavy lines approximately perpendicular to the

fibril axis (Figure 4.9a). In loaded tendons the D-periods appeared to straighten,

assuming a skewed orientation to the respective fibril axes (Figure 4.9b).

Similar to the observed changes in fibril d-period elongation, collagen fibril diame-

ter apparently decreased with applied load. The mean fibril diameter for the control

group with 0N load and 0% strain was found to be to 193.6 ± 35.5 nm. At 5%

applied strain the diameter was statistically unchanged (195.6 ± 32.2 nm), but was

significantly lower at 10% applied strain (175.3 ± 27.7 nm, p < 0.05). Comparison

between all groups of target strain level did not indicate different characteristics

with respect to fibril spacing, intertwined fibrils or broken fibrils. Analyzing single

fibrils within an individual tendon indicated no correlation between d-period length

and fibril diameter; fibril diameter was uniformly distributed over the d-period range

(data not shown).

Discussion

While many studies have investigated tendon structure-function at the ultrascale,

much remains unclear regarding the precise ultrastructural mechanisms that under-

lie the versatile biomechanical behaviors of tendon such as elastic energy storage

and recovery after creep [3]. These mechanisms include straining, relaxation, creep,

and kinematic reorientation of loaded collagen fibrils in the extracellular matrix. Al-

though collagen fibril morphology and non-collagen matrix composition are widely

believed to dictate macroscopic behavior [10, 34, 35, 11, 36, 19], how these ultra-

structural aspects translate to tissue function is poorly understood. This is due in

75

Material level

Figure 4.8: Probability distribution of the collagen fibril d-period length as a func-tion of the measured strain on the tendon. The d-period length is increas-ing with increasing total strain of the tendon, whereas the variability ofthe d-period length is decreasing with increasing strain.

Figure 4.9: Height AFM images of collagen fibrils taken in contact mode for (A) anunloaded tendon, and (B) a tendon stretched to 10% strain. The z-rangeis indicated by grayscale bar with values in nm.

76

4.2 Collagen fibrils’ role in tendons behavior

Figure 4.10: Plot of the applied tension for the bulk tendon, the measured d-periodlength of the collagen fibrils and the mean collagen fibril diameter as afunction of the target strain. At 10% strain, the increase in the d-periodlength and the decrease in fibril diameter is statistically significant (*indicates a p-value < 0.05). No significant difference was found betweenthe 0% and 5% strain groups.

part to inherent methodological limitations of experimental approaches that have

been applied to quantifying collagen fibril strains under load (scattering x-ray spec-

troscopy [7]; scanning electron microscopy [20]; longitudinal TEM [21, 22]; isolated

fibril studies using AFM [16]); While each method has yielded valuable but partial

insight into collagen fibril mechanics, none of these methods has proven capable to

characterize heterogeneity in collagen fibril load distribution in the tendon extracel-

lular matrix. Available methods therefore offer little to the study of collagen fibril

load sharing.

In this study we attempted to implement AFM to visualize individual collagen

fibrils in murine Achilles tendons under load. We expected that the high morphome-

tric accuracy of AFM imaging, combined with an experimental approach permitting

77

Material level

a “snapshot” of the strained collagen fibrils within their network could yield unique

insight into the nature of collagen fibril load bearing. To enable this characteriza-

tion, we quantified the periodic banding and diameters of over 200 collagen fibrils

in loaded and unloaded tendon, and analyzed them to respectively assess collagen

fibril straining and thinning.

We explored several hypotheses regarding an expected mechanical loading hetero-

geneity in the collagen network. For instance, we hypothesized that a multi-modal

distribution of collagen fibril strains would be observed, and that this could be fur-

ther related as a function of fibril diameter. Numerous authors (including ourselves)

have conjectured that the bimodal distribution of large and small diameter fibrils

may respectively enable load and creep resistance [37, 9, 25, 26, 38]. This hypothesis

was not born out by our data, which indicated that the collagen network stretches in

a very uniform manner, both locally and across various anatomic tendon subregions,

without obvious differences in mechanical strains between neighboring fibrils or be-

tween fibrils of different diameters. In this sense our results were rather unexpected,

and indicate that a refined view of diameter biased fibril recruitment is required.

Based on previous reports [3, 7, 5], the expected behavior of the collagen fibrils

was of a statistically longer d-period in the loaded tendon compared to the unloaded

tendons. Our study confirms these findings. We further confirm the reported dis-

crepancy between applied macroscopic tendon strain and the resultant collagen fibril

strain where overall strain of the tendon fiber appeared always larger than the strain

in individual fibrils [8, 24, 7]. In the “toe region” of the material curve, tendon strain

is characterized by no corresponding collagen strain. This implies that the mechan-

ical resistance derives mostly from inter-fibril movements within the matrix, and

that strain of the collagen fibrils itself is a likely a protective mechanism against

overstrain. Fratzl et al attributed this behavior as the elongation of the macroscopic

“crimp” that disappears after approximately 5% fiber strain [8]. After 5% strain

the entire structure is then under load and the fibrils begin to actively bear incre-

mentally applied load. For strains larger than 5%, the d-period elongation has been

reported to contribute approximately 50% of the total measured fiber elongation [7].

Our own data (Figure 4.2) also indicate that fibrils start to significantly elongate

only above 5% tendon strain, but that this increase in d-period strain amounted to

only approximately 20% of the incrementally applied tendon strain. We attribute

this difference to the fact that the tendons we assessed presented a more complex

structure than those assessed in earlier studies on isolated tendon fascicles [3, 39].

Additional work will be required to clearly establish how the differences in mechan-

ical strain between whole tendon, tendon fascicles/fibers, and collagen fibrils relate

78

4.2 Collagen fibrils’ role in tendons behavior

to the structural organization of the tendon.

Similar to the observed trends in fibril elongation, collagen fibril diameters also

appeared unchanged at 5% strain and then statistically differed at 10% applied

tendon strain. The observed decrease in mean fibril diameter corroborates an earlier

study indicating smaller diameter fibrils in loaded tendons [25].

Although this study offers additional insight into the distribution of load among

collagen fibrils, it does have some limitations. First, because destructive process-

ing steps were required to gain visual access to the tendon core, AFM imaging of

native (unfixed) tendon structures was not possible. Glutaraldhyde fixation was

thus used to preserve native morphology of the mechanically loaded collagen fibrils.

Glutaraldehyde fixation has long been relied upon as a fixative in morphological

studies of collagen using electron microscopy (e.g. [40]), and this approach has more

recently been utilized to fix native collagen morphology under load [41, 42, 30]. We

thus believe that 8 hours of glutaraldehyde fixation of fresh (native) tendons under

load adequately captured and preserved the native morphology of the loaded colla-

gen fibrils. It should be noted that the amount of creep occurring over the course of

chemical fixation was uncontrolled [30]. However, the majority of creep strain was

small relative to the applied target strain (Figure 4.5), occurred mostly within the

first ten minutes of fixation, and was generally consistent from sample to sample.

While time dependent effects are secondary to the mechanical response of the fibers

and fibril within the first 20 seconds of an applied load [3], creep loading and uncon-

trolled dynamics of chemical fixation may have nonetheless influenced the resultant

fibril morphology. While we made an effort to minimize such effects by mechanical

preconditioning, quantifying effects of and differences between creep and relaxation

remain ground for future work.

As a separate limitation, the preparation of high quality slices for AFM analysis

was experimentally challenging. Before sectioning, all samples were embedded in

a medium containing polyvinyl alcohol which is known to form thin films. Some

samples, or sample subregions, showed poor image quality (i.e. smoother “edges”

of the fibrils), possibly due to surface residues, and these had to be excluded for

morphometric analysis. While we have confidence in the fidelity of the image data

we did not exclude the possibility that film residues may have adversely affected

morphometric accuracy of the subsequent d-period and diameter measures.

In conclusion, we have presented a novel and potentially useful method for in-

vestigating the mechanical response of collagen fibrils in their parent tissue. Using

this method, we have produced visual, quantitative evidence that mechanical loads

in the murine Achilles tendon are approximately uniformly distributed among the

79

Material level

resident collagen fibrils, apparently independent of collagen fibril diameter. In pro-

viding an accurate analysis of loaded collagen fibril strains in response to applied

stress or strain, this method may provide a superior means of investigating complex

structure-function hypotheses in tendon.

Acknowledgements

This work was supported through the BEST Bioengineering Cluster at ETH Zurich

(Switzerland) and the EU grant LHSB-CT-2003-503161 (Genostem).

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84

4.3 Tendon glycosaminoglycan proteoglycan

sidechains promote collagen fibril sliding — AFM

observations at the nanoscale

S. Rigozzi1, A. Stemmer2, R. Muller1, and J.G. Snedeker1,3

1Institute for Biomechanics, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich,

Switzerland

2Nanotechnology Group, ETH Zurich, Tannenstrasse 3, 8092 Zurich, Switzerland

3Orthopedic Research Laboratory, University of Zurich, Balgrist, Forchstrasse 340,

8008 Zurich, Switzerland

submitted to:

Journal of Structural Biology

Nov 2011

Abstract:

The extracellular matrix of tendon is mainly composed of discontinuous Type-I

collagen fibrils and small leucine rich proteoglycans (PG). Macroscopic tendon

behaviors like stiffness and strength are determined by the ultrastructural arrange-

ment of these components. When a tendon is submitted to load, the collagen

fibrils both elongate and slide relative to their neighboring fibrils. The role of PG

glycosaminoglycan (GAG) side chains in mediating inter-fibril load sharing remains

controversial, with competing structure-function theories suggesting that PGs may

mechanically couple neighboring collagen fibrils (cross-linking them to facilitate

fibril stretch) or alternatively isolating them (promoting fibril gliding). In this study

we sought to clarify the functional role of GAGs in tensile tendon mechanics by

directly investigating the mechanical response of individual collagen fibrils within

their collagen network in both native and GAG depleted tendons. A control group of

Achilles tendons from adult mice was compared with tendons in which GAGs were

enzymatically depleted using chondroitinase ABC. Tendons were loaded to specific

target strains, chemically fixed under constant load, and later sectioned for morpho-

logical analysis by an atomic force microscope (AFM). Increases in periodic banding

Material level

of the collagen fibrils (d-period) or decreases in fibril diameter were considered

to be representative of collagen fibril elongation and the mechanical contribution

of GAGs at the ultrascale was quantified on this basis. At high levels of applied

tendon strain (10%), GAG depleted tendons showed increased collagen stretch (less

fibril sliding). We conclude that the hydrophilic GAGs seem thus not to act as

mechanical cross-links but rather act to promote collagen fibril sliding under tension.

Keywords:

Tendon, Collagen proteoglycan interaction, atomic force microscopy, molecular me-

chanics, glycosaminoglycans

Introduction

Collagen and proteoglycans (PGs) are the principal constituents of tendon by dry

weight [1]. At the ultrascale, tendon collagen fibrils are structural elements with

diameters between 10 and 400 nm that can self-assemble until reaching hundreds of

micrometers in length [2]. The fibrils are typically aligned to the functional axis of

tendon, bringing tensile stiffness and strength to the tissue [3]. The small leucine

rich PGs in tendon (primarily decorin and biglycan) are known to regulate the mod-

ulation of growth factor activity and to facilitate organization of the extracellular

matrix during fibrillogenesis (ECM) architecture [?, 4, 5]. PGs are constituted of a

core protein to which one or more secondary glycosaminoglycan (GAG) sidechains

are covalently bonded. The GAGs found in tendons are typically dermatan sulfate

(DS), chondroitin sulfate (CS) and keratan sulfate (KS). These hydrophilic chains

play a large role in osmotic balancing of hydrostatic pressures, and thus provide

tendon with mechanical resistance to compressive loads [6, 7]. On the other hand,

the mechanical contribution of decorin other PGs under tensile load remains a point

of contention. Based on experimental observations, numerous researchers have sug-

gested that PGs may mechanically link adjacent collagen fibrils and thus play a

direct role in inter-fibril load sharing [8, 9, 10, 11, 12, 4, 13]. Theoretical models

have been further introduced that verify the mechanical plausibility of this theory

[14, 15, 16]. These models conceptualize tendon as a composite material of stiff fibers

embedded within a softer matrix, with a remarkably large cumulative mechanical

effect of weak individual PG binding forces. This substantial cumulative effect stems

from the extremely large length to diameter ratios of collagen fibrils, which provides

an enormous amount of surface area for inter-fibril forces to act. These models thus

adopt the classic theory of “shear-lag” which assumes that shear forces distributed

86

4.3 PGs’ mechanical role in tendon behavior

along the entire length of the fiber act through the surrounding matrix to transfer

loads between adjacent fibers [17, 18, 19].

Despite the attractiveness of the ultrastructure-function theory of PG mediated

collagen load sharing, experimental results on murine tendon from decorin and bigly-

can knock-out models have inconclusively linked absence of these PGs with altered

tendon mechanical properties [11]. Similarly disputing this theory, GAG content

and mechanical properties were found to be inversely correlated when comparing

Achilles tendons between strains of inbred mice [20]. Finally, more direct studies

comparing native tendon with GAG-depleted tendon (enzymatically depleted using

chondroitinase) indicated minimal, if any, PG contribution to tensile properties of

the tissue [21, 14, 22]. Similar enzymatic GAG depletion studies in ligament rein-

force these findings [23, 24]. These studies at the tissue level thus bring into doubt

the primacy of PGs in mediating collagen load sharing.

The key difference between those studies supporting PG mediated collagen load

sharing and those studies rejecting it, is the size scale of the experiments themselves.

Nearly all of the studies supporting the theory have been based on observational

(semi-quantitative) electron microscopy imaging studies that focused exclusively on

collagen-proteoglycan interactions and treated the tendon as a mechanical contin-

uum at this level. On the other hand, the cited studies that collectively refute PG

mediated collagen load sharing all employ experiments at the tissue level. While

tissue level experiments provide relevant insight into the impact of PGs at the or-

ganism level, such experiments include potentially confounding effects from each of

the intervening levels of structural hierarchy [25, 26, 27]. To bridge this gap, we

present here what we believe to be the first quantitative ultrascale study of collagen

fibril load bearing (and sharing) as a function of PG GAG content.

In the present study we utilize a novel method for characterizing effects of me-

chanical load on in situ single collagen fibril morphology [28]. This method employs

atomic force microscopy (AFM) to quantify load dependent changes in fibril banding

periodicity (d-period) and diameter, which both can be used as a measure of colla-

gen fibril stretch [3, 29, 30, 28]. We used this direct imaging approach to test the

hypothesis that enzymatic depletion of GAG, PG sidechains, would alter collagen

fibril load distributions. More specifically, we presumed that tendon inter-fibril load

sharing is dependent on PG cross-linking, and that disrupting GAG interconnec-

tions would lead to decreased transmission of collagen shear forces under load. At

the ultrascale, this removal of fibril-coupling should translate to increased collagen

fibril sliding and decreased collagen stretch compared to native tendon at the same

applied tissue strain (see Figure 4.11).

87

Material level

The aim of this study was therefore to clarify the still controversial role of PGs

in collagen fibril load sharing. We implement a direct functional imaging approach

that combines mechanical loading of whole tendon with ultrascale imaging analysis

using AFM. Loaded collagen fibril morphology of native tendons and GAG-depleted

tendons, specifically d-period and fibril diameter, were quantified and compared.

According to our hypothesis, a statistically shorter d-period in GAG depleted groups

would support a direct contribution of PGs to tensile tendon mechanics.

Figure 4.11: Schema of the mechanical behavior of collagen fibrils connected by PGcross-links. (top) Fibrils in a high cross-linked network slide and elon-gate homogeneously to bear the load. (bottom) For GAG-depletedtendons, we hypothesize that the fibrils are loosely coupled showing alower fibril strain and a higher inter-fibril gliding than a native tendon.If the fibrils are tightly coupled, the total fibril strain would increaseand the the inter-fibril gliding decreases.

88

4.3 PGs’ mechanical role in tendon behavior

Materials and Methods

Sample preparation

All animal experiments were reviewed and approved by local and state authorities

(Kantonales Veterinaramt Zurich, Zurich, Switzerland). The soleus tendons were

harvested from the Achilles tendons of female 19-week-old C57BL/6 inbred (wild

type) mice. A control group of 15 native tendons (left leg) was incubated for 12h

at 37◦C in Tris Hydrochloride 0.1M Sodium Acetate 0.1M buffer (pH 8), while a

GAG-depleted group of 15 tendons (right leg) was incubated for 12h in Tris buffer

+ 0.15U/ml chondroitinase ABC (ChABC, Sigma-Aldrich, Switzerland), pH 8; in

order to reduce the number of PG GAG sidechains [21, 31, 22].

The tendons of each group were divided into 3 subgroups each of 5 tendons that

were then stretched to different target strains (0%, 5% and 15% nominal strain).

The tendons were held under constant force and fixed with glutaraldehyde as pre-

viously described [28] (Figure 4.12a). To separate potentially confounding effects of

dehydration associated with GAG depletion, two additional groups from both native

and the digested tendons were measured at 0% strain. In these experiments, the

control group consisted of tendons that were incubated in buffer (with and without

ChABC) and chemically fixed in glutaraldehyde, the dehydrated group included

tendons that were dissected, incubated in buffer (with and without ChABC) and

then successively soaked for 1,5h in a freshly prepared solution of 8% PEG in buffer,

in order to dehydrate the tissue [21]. The relative water gain/loss of these tendons

during the different preparation steps was recorded by measuring the wet weight of

tendons after each processing step. The tendons were then fixed at 0% strain in

1.25% glutaraldehyde using the protocol described above.

GAG analysis

After weighing, two additional groups of 5 tendons were treated according to the

native and GAG depleted protocols, and were characterized for GAG content using

the Farndale method [32]. Tendons were digested for 16h with 500 U/ml papain

solution in buffer (0.1M disodium hydrogen phosphate, 0.01M EDTA disodium salt,

14.4mM L-cysteine) at 60◦C. GAG content was determined spectrophotometrically

(Cary 50, Varian, Zug, Switzerland) at 525 nm following binding to dimethylmethy-

lene blue dye (DMMB) using CS as standard.

89

Material level

Figure 4.12: Experimental set-up of the tendon preparation and analysis. A) Thetendon is clamped in a tensile test machine under constant load to reacha defined target strain. The chamber is filled with 1.25% glutaraldehydeto fix the tendon chemically while submitted to constant load. B)Representative AFM height image of a tendon fascicle showing collagenfibrils characterized by the repeated gaps forming the d-period. C) Twodimensional height profile of the black line in the image B, and D) resultgraph obtained after Fourier function transform analysis of the two-dimensional profile from Figure 4.12c.The highest peak corresponds tothe inverse of the length of the most frequent periodicity.

90

4.3 PGs’ mechanical role in tendon behavior

Image analysis

After chemical fixation all tendons were embedded at -20◦C in Tissue-Tek (Tissue-

Tek, 4583 Compound, Sakura Finetek Europe, Zoeterwoude, Netherlands) for at

least 12h. Each frozen tendon was then cut with a cryostatic microtome (SLEE,

Mainz, Germany) at -20◦C into three 20 µm thick slices and left to dry on a

polylysine-coated glass slide (SuperFrost, Menzel-Glaser, Braunschweig, Germany)

at 60◦C for another 12h.

AFM experiments were then performed with an Asylum Research MFP-3D-SA

(Germany). Images of collagen fibril topography were acquired in contact mode

at a scanning rate of 0.5 Hz in air on the dehydrated thin-sectioned samples (Fig-

ure 4.12b). All measurements were carried out at room temperature. Titanium and

platinum-coated, silicon AFM tips on triangular cantilever with a nominal spring

constant of 2.35 N/m (NSC11/Ti-Pt; MikroMasch, Madrid, Spain) were used. In

each slide five region of interest (ROI) were analyzed, which contained between 5

and 10 fibrils on the surface that were randomly identified and measured.

The d-period of individual collagen fibrils within their collagen network was mea-

sured as described earlier [28]. Briefly, analyzed fibrils in each image were charac-

terized by a two-dimensional height profile (Figure 4.12c). A Fourier transform was

then applied to calculate the inverse of the d-period length corresponding to the

highest peak in Figure 4.12d. For each analyzed fibril, diameter was measured from

the two-dimensional height profile of taken approximately perpendicular to the fibril

long axis.

Statistical analysis

Measured wet-weights for the control and treated groups (with and without ChABC)

were tested with Bonferroni correction for multiple comparison with significance level

set at p-value < 0.05. For the fibril structural quantification, the d-period lengths

and diameters of analyzed collagen fibrils were pooled together within experimental

groups according to target strain. After applying a Lilliefors test to verify whether

distributions could be approximated by a normal distribution, ANOVA tests were

used to test for differences in d-period length and diameter between treated and

control groups at similar applied strain levels. A p-value < 0.05 was considered to

be significant.

91

Material level

Results

Assays of GAG content in native and ChABC treated groups indicated a 60% reduc-

tion in GAG content in the treated tendons (2.6 ± 1.1 µg/mg vs. 6.1 ± 0.4 µg/mg,

p-value <0.05). The effects of incubation (swelling and the relative dehydrating ef-

fect of GAG depletion) on d-period length were analyzed at 0% strain. Two groups

of tendons were dehydrated with PEG according to previous studies [21] showing

a statistical decrease in water content of about 15% for both native and digested

groups (p<0.05). At the ultrascale, no significant differences in d-period lengths

were found after incubation and dehydration (65.61 ± 1.61 nm) compared to native

controls (65.28 ± 1.32 nm) (p=0.08). Similarly, no differences were observed in D-

period lengths of GAG-depleted tendons at 0% applied strain as a function of PEG

dehydration (65.00 ± 1.89 nm incubation only; 65.75 ± 2.26 nm incubation and

PEG dehydration; p=0.22). Collagen fibril diameter was not apparently affected by

GAG-depletion, and neither control nor GAG-depleted tendon fibril diameters were

significantly affected by swelling (Table 4.5, Figure 4.13).

After verifying against potential confounding effects of dehydration due to the

treatment protocol, we probed for differences in mechanical response in the native

and the digested groups in terms of fibril d-period and diameter (Figure 4.14).

No differences in collagen morphology were detectable at applied tendon strains

corresponding to the “heel” region of the material curve (5%) for either GAG-

depleted or native tendons. For higher tendon strains corresponding to the linear

region of the material curve (15%), there was a significantly increased d-period length

in both groups compared to corresponding low-strain groups. Moreover, d-period

length was found to be statistically longer in GAG-depleted tendons compared to

native tendons at similar target strains (p <0.05). Measured collagen fibril diameter

was 19% lower in highly strained tendons (15%) than unstrained controls (p <0.005),

but with no detectable difference between native and GAG digested groups at similar

target strains. Comparison of d-period length and fibril diameter distributions in

native and GAG-depleted tendons indicated that GAG-depleted fibrils had generally

more morphological variability than corresponding controls.

Discussion

Collagen load bearing is by far the most important factor in tendon mechanics

[26, 33, 12, 27]. At the ultrascale, tendon elongation is enabled by the mechanisms

of collagen fibril sliding and collagen fibril elongation that occur simultaneously and

in a critical balance. Tendon proteoglycans have been implicated in regulating this

92

4.3 PGs’ mechanical role in tendon behavior

Table 4.5: Incubation effect on Type I collagen’s d-period and fibril diameter at0% strain. The control group was incubated in PBS and successivelychemically fixed, the dehydrated group was incubated in PBS, in PEGand successively chemically fixed and the digested group was incubatedin PBS with ChABC and chemically fixed.

Group Control Dehydrated Digested Digested and dehydrated

D-period (nm) 65.28±1.32 65.61±1.61 65.00±1.89 65.75±2.26

Diameter (nm) 204.59±30.76 209.07±32.10 195.35±36.64 194.96±38.27

balance [26, 34]. Given their ubiquity and regular spacing (64-68 nm intervals) along

the sides of collagen fibrils [13], small leucine rich PGs such as decorin and biglycan

have been suggested to possibly transfer forces between collagen fibrils by mechan-

ically coupling them in the structure [8, 10, 15, 16]. Our previous quantitative

analysis of structure and function have indicated that the mechanical contribution

of PGs to tendon tensile mechanics is probably minimal, but potentially complex

[21, 22, 28]. In the present study, we sought to more directly investigate how GAG

sidechains of small leucine rich proteoglycans may mediate collagen fibril load shar-

ing.

To this end, we employed a quantitative ultrascale analysis of collagen fibril me-

chanics using a recently developed AFM-based functional imaging approach [28].

Tendons were stretched to different target strains and their structural response at

the ultrascale was quantified by analyzing collagen fibril d-period length [35] and

diameter [36]. This functional imaging approach is able to capture a “snapshot” of

the collagen fibrils within the network, but is limited in some aspects. The effective

time that glutaraldehyde needs to totally fix the tendon under tension could not be

precisely defined. Although consistent between the samples, some creep that might

have been occurred in the structure could not be defined. Moreover, to prepare the

structure slices for the AFM observation, all tendons were embedded in a medium

containing polyvinyl alcohol, which might form an additional thin film on the struc-

ture that lead sometimes to image artifacts: reduction of the structural details or

surface residues. We used this approach to elucidate mechanical contribution of

GAGs on collagen mechanical load transmission by enzymatic GAG depletion by

ChABC. We decided to use ChABC instead of knock-out mice, because the lack

of specific genes that assure a lower PG concentration do not ensures that other

processes compensate this lost to restore the same function in the body [11, 37].

Although ChABC digestion might affect the tendon structure by collagen fibrils

swelling [31] or alteration of other components [38, 23], we attempted to preclude

93

Material level

Figure 4.13: Representative transmission electron micrograph (A,C) and AFMheight images (B,D) of the longitudinal section of a native control (A,B), and a dehydrated Achilles tendon (C, D). The plot displays themean d-period length and collagen diameter measured for native anddigested tendons that were normally incubated in PBS (control group),or successively dehydrated with PEG (dehydrated group).

confounding morphological changes associated with incubation, dehydration or en-

zymatic digestion by ChABC, with visual observation at 0% strain indicating no

measurable artifacts due to the experimental protocols (Table 4.5). The morpholog-

ical differences we observed between treatment groups in relation to the mechanical

response were therefore attributed to functional differences due to GAG content

(here measured to be 60% lower for ChABC treated tendons).

Consistent with our previous AFM functional imaging study of loaded tendon

collagen structures, native d-period elongated in accordance with applied tendon

strain (Figure 4.14), while collagen fibril diameter decreased at higher strains [28].

The GAG depleted group showed similar trends, with longer d-periods and smaller

collagen fibril diameters at 15% applied strain compared to 0% and 5% strain groups

(p-value <0.005 with 0% strain and p-value <0.05 with 5% strain). In both native

and GAG-depleted tendons , no d-period lengthening was observed until 5% applied

strain, which at the macroscale corresponds to loss of collagen fiber crimping [30].

However, a direct comparison between native and GAG-depleted samples at 15%

94

4.3 PGs’ mechanical role in tendon behavior

Figure 4.14: Mean fibril diameter and d-period length versus total measured targetstrain in the tendon for native and digested tendons. In the nativetendon the d-period length is statistically longer for 15% in comparisonto the 5% strain group. The digested tendon shows a regular increasein the d-period length that is significant at 15% strain with both the0% and 5% target strain group. At 15% strain a statistical differenceis showed between the native and digested tendons. For both nativeand digested tendon group the diameter is gradually decreasing and isstatistically smaller over 5%.

applied strain showed higher levels of fibril strains in the GAG digested tendons

(p-value <0.05). Simultaneously, the collagen fibrils diameter of the digested group

was smaller than in native tendons, although not significantly. It thus appears that

GAG content in native tendon is related to lower collagen strains (reduced collagen

fibril stretching, with an implied corresponding increase in collagen fibril sliding).

This finding is inconsistent with the concept that GAG sidechains of PGs act as

mechanical cross-links that transfer shear force between neighboring collagen fibrils

in the collagen network. That tendon behavior depends on GAG mediated shear

transfer between collagen fibrils is directly contradicted by the fact that removing

GAG “connectivity” between the fibrils and matrix leads to a higher fibril strains. In

contrast, these data suggest that the highly hydrophilic GAGs act to mechanically

isolate individual fibrils, facilitating sliding and perhaps “protecting” collagen fibrils

from overstrain. This conclusion is further supported by our earlier observations in

murine Achilles tendons positively relating higher collagen fibril surface area and

increased GAG concentration with a reduced mechanical stiffness [20].

These findings thus complement (and perhaps complete) our previous studies

95

Material level

using mechanical testing on GAG-depleted tendons to demonstrate that tensile me-

chanics of native tendon is not heavily dependent on GAG content [14, 21, 22, 20, 28].

The current study does however introduce a subtle, and perhaps important, mechan-

ical role that GAGs play in promoting fibril sliding. This may play a key role in

“slow” viscoelastic mechanical processes such as creep or relaxation [21], and GAGs

could plausibly enable creep recovery under cellular tension of the matrix. In any

case, GAGs play an essential role in collagen fibrillogenesis and are essential to

the attainment of proper mechanical properties in tendon development and heal-

ing [39, 40]. Further, aberrant levels of PGs and their associated GAG chains is

correlated to poor functional recovery after injury. So although we can now con-

fidently conclude that tendon PGs do not play a “collagen cross-linker” role, the

small leucine rich proteoglycans continue to lie at the center of tendon structure

and function.

Acknowledgements

This work was supported through the BEST Bioengineering Cluster at ETH Zurich

(Switzerland) and the EU grant LHSB-CT-2003-503161 (Genostem).

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100

Chapter 5

Synthesis

Tendon is a passive connective tissue that transfers muscle forces to the skeleton,

enabling minimal energy loss during load transfer while allowing enough extension

to prevent injury in the compliant muscle connected to the rigid bone [1]. Although

the gross mechanical characteristics of tendon are well documented, the role of the

structure in facilitating and controlling the mechanical behavior is less clear [2].

Tendon injuries are associated with enormous costs in the work place [3] and account

for 30% to 50% of all sports injuries [4, 5]. Despite decades of research and increasing

clinical attention to these injuries, their clinical outcome remains unpredictable [6,

7, 8, 6, 9]. A comprehensive understanding of the physiological and biomechanical

processes that underlie tendon function and pathology is essential to improve clinical

diagnosis and treatment of tendon pathology.

Collagen structure-function

The mechanical integrity of tendon was historically attributed to collagen fibrils that

are predominantly arranged into longitudinal fibrils with diameters ranging between

20 and 400 nm. Fibrils are the stiffer component of tendon located in the extracel-

lular matrix (ECM) in close proximity to their neighbors, with radial separations

of several orders of magnitude less than fibril length. Several studies characterized

the fibril morphology and mechanical properties [10, 11, 12, 13, 14]. Others com-

pared the tendon to a composite material in an effort to better understand how the

collagen influences the overall mechanical response [15, 16, 17, 18]. In general, all

these studies focused on one specific hierarchical level and its mechanical response.

More recent studies that focus on the collagen structure-function are considering the

phenomena that occur simultaneously at different scale levels of tendon throughout

the hierarchy [19, 2, 20]. When a tendon is under load there are several phenomena

that occur simultaneously at different scale levels. The collagen molecules extend

and there is a sliding between the collagen fibrils and fibers as well [2]. The sliding

Synthesis

occurring in the structure is probably controlled by the surrounding PG matrix,

which is the other main component of tendon [21, 22].

Potential role of proteoglycans

PGs were widely studied over the last decades; they are thought to principally trans-

fer force between the fibrils under tension [23], or to be responsible for the viscoelastic

nature of tendon as a consequence to their high affinity with water [24]. Theoretical

models have been introduced on the basis that PGs act as elastic bonds between

the collagen fibrils that transfer forces between them [25, 26]. Experimentally, Liao

supported this theory by showing the skewness angle of PGs between the fibrils

changes with applied load [23], however it is unclear whether this change is directly

caused by the increasing load or is just a reorganization of the PGs. Conflicting

studies found no influence in the mechanical response that can be attributed to PGs

[27, 28, 29]. Other studies focused on the viscoelastic properties of the tendon to

model its mechanical response [30, 31]. Microstructural modeling studies suggested

that the inter-fibrillar matrix is important for controlling the viscoelastic behavior

[32, 33]. This latter proposal is supported by experimental studies which indicated

sliding between fibers during quasi-static tissue loading [21, 2].

Into the big picture

Despite a dearth of quantitative evidence regarding the precise roles of the ultra-

structural tendon components, the tendon structure-function relationship has been

widely speculated upon. This relationship has been partly supported by experi-

mental studies that have indicated correlations between tendon failure strength and

collagen content [10, 14, 34] as well as correlations between collagen fibril number

and size with tendon stiffness [35, 36]. However, many of these experiments have

yielded conflicting and/or counterintuitive results indicating that basic assumptions

regarding how ultrastructure relates to function need to be reevaluated.

This thesis aimed to illuminate some of these aspects by providing a new approach

to analyze the influence of the tendon mechanical components in the whole tensile

response. In this work, tendons were mechanically investigated at the tissue-organ

level in relation to the PG concentration and the collagen fibril diameter. Their

structure was further analyzed at the microscale in order to elucidate the mechanical

contribution at a smaller hierarchical level.

The major aim of Chapter 3 was to examine the possible functional role of PGs

and their related secondary chain — i.e. the glycosaminoglycans (GAGs) — in the

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tensile mechanics of murine Achilles tendon. Previous studies showed that the con-

centration of PG is not equally distributed in the tendon [37]. In Achilles tendon,

the tensile region contains mainly small PGs, while the compressed region towards

the bone insertion have more aggrecans [38]. Thus, a novel experimental set-up was

first developed and validated in order to investigate the regional variation of the

tendon elongation in tension depending on PG concentration. The total tendon me-

chanical response was analyzed mechanically with a universal tensile machine that

recorded elongation under load; simultaneously frames of the posterior view of the

tendon were collected with an external camera. The optical acquisition of the me-

chanical response of the tendon was used to regionally analyze three regions equally

distributed on the longitudinal axis. From the recorded video it was possible to see

that murine Achilles tendons were formed from intertwined primary fiber bundles

that twisted in a coordinated fashion under load. These bundles had unique func-

tional axes, and were surrounded and interconnected by fasciae. The combination

of both the mechanical and the optical measuring systems of the overall mechanical

response and the regional elongation of the Achilles tendon, showed that tendons do

not behave homogeneously along their tension axis. Furthermore, a digestion of the

GAGs in the tendon induced a softening at the bone insertion, which has naturally

the highest concentration of PG [39]. Variations in PG concentration did not alter

the intrinsic mechanical response in the midsubstance of the tendon (the tensile

region), as supported by later studies investigating the mechanical influence of PG

concentration in soft tissues [28, 27]. This first study emphasized the complicated

role of PG in tension, and then in the following chapter an investigation at a lower

hierarchical scale was made in order to elucidate the phenomena occurring further

in the structure.

In Chapter 4.1 the local mechanical analysis of the tendon mechanics was related

to detailed collagen fibril morphology of two inbred mouse strains. In tensile load

the mouse strain exhibiting the higher E-modulus (C3H) was found to have collagen

fibrils with a larger mean diameter and smaller specific fibril surface (the surface

surrounding the fibril that is in contact with the non-collagenous part of the ECM).

Furthermore, GAG analysis in both groups showed that the stiffer-tendon mouse

group (C3H) had a lower GAG concentration in the tissue. This result concurs with

the well-known property of PGs to modulate the formation and final size of collagen

fibrils [40, 41], and other studies that show large fibril size in a lower GAG con-

centration milieu. In contrast to previous studies, this particular structure-function

analysis of tendons considered the functional relevance of collagen fibril morphology

and GAG content, thus focusing on the potential interplay between collagen and

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Synthesis

PGs. The goal was to better understand the mechanical contributions of these two

components that are known to influence each other during tissue formation [42, 40].

This study attempted to correlate the collagen fibril morphology to the tendon me-

chanical response. If the tendon is compared to a composite material made of stiff

fibers embedded within a softer matrix, from a simple equation of the composite

material (see equation 2.2), for equal fibril and matrix E-modulus, the fibril area

fraction should directly influence the total E-modulus. The results found in this

study show that there is not a significant correlation between the fibril density and

the tendon E-modulus. However, the E-modulus was found to be significantly larger

for a smaller specific fibril surface (the surrounding area of the fibril). Moreover the

GAG concentration in the tendon was positively correlated with the specific fibril

surface, which is believed to the the contact area of the PG chains [33]. GAG con-

tent could be related to promoted fibril sliding, which one would expect to reduce

E-modulus [24]. This statistical difference in the GAG concentration for different

E-moduli re-opened the question of the GAG’s role in mechanics since the two first

studies at both tissue-organ and material level gave conflicting results. This differ-

ence might be solely a consequence of fibril morphology since they constitute 60% of

tendon composition. Another plausible explanation of this difference could be that

the chemical digestion of the GAGs altered the structure and increased the biolog-

ical variability in such a way as to reduce the mechanical capacity of the tendon.

A complementary investigation at this scale level was therefore critical to elucidate

this issue.

With this last statement, Chapter 4.2 and 4.3 focused on the interactions be-

tween the tendon components in their native network at the nanoscale with the

overall mechanical response. A new experimental approach combining macroscopic

mechanical loading of tendon with a morphometric ultrascale assessment of longi-

tudinal and cross-sectional collagen fibril deformations was developed and validated

in Chapter 4.2. Complex anatomical structures composed of intertwined fascicles

are constantly modeled [43, 44] in efforts to understand the interaction between

these structures under load. The motivation for this analysis was the lack of exper-

imental studies that relate the nature by which collagen fibrils interact within their

network to accommodate applied tendon strains. The collagen molecule extension

was monitored by assessing d-period length and diameter changes with atomic force

microscopy (AFM). The innovation of this method was the ability to mechanically

load a entire tendon and to analyze how the collagen fibrils behave in their net-

work that was not altered. The measurements showed that the mean collagen fibril

elongation measured in the network is smaller than the whole strain displayed by

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Synthesis

the tendon. The new method was consistent with published reports performed with

different techniques [45, 2, 21], which also report a discrepancy between the total

strain measured at the macroscale and at the ultrascale. However, the analyses in

these studies were made with isolated fascicle or fibers and were limited to discerning

in situ strains in individual collagen fibers. As soon as a tissue structure is altered

to isolate a part of it, all the network movement and the influence of the neighbors

can not be measured. The advantage of the approach presented in Chapter 4.2 is

that the structure was not altered before the mechanical response occurred. Achilles

tendons were stretched and chemically fixed with their native structure remaining

untouched, so the whole mechanical response was the sum of contribution of each

component. With this new approach the influence of the PG chain to the mechanical

response was measured in the following study.

Finally, in Chapter 4.3 the collagen fibril mechanical response in tension in re-

lation to different PG concentration was characterized with the AFM. With the

novel method developed and described in the previous subchapter the mechanical

contribution of PGs at the ultrascale was quantified by comparing the difference in

the d-period length and the collagen diameter between native and GAG-depleted

tendon. No change in collagen diameter was observed between the groups, however

the fibrils from the group with a lower concentration of GAG presented a greater

elongation for equal tendon strain. This implies that in tendons with a lower GAG

concentration the collagen fibrils were more stressed under tensile load. This sur-

prising outcome was in contrast with a common assumption that the PGs are linked

to the collagen fibrils and transfer the load between them [46]. Such a network of

cross-links could facilitate the load transfer in the matrix and provide stiffness to

the tissue if they act simultaneously. A depletion of GAG was originally thought as

a malfunctioning network where not all the fibrils can be loaded as schematized in

Figure 4.11. However, in fact a lack of GAG resulted in a higher strain in the fibrils

implying a highly coupled structure that bears all the load. Since PGs are highly

hydrophilic [42], this suggests that the PGs are implied in the mechanical response

of the tendon as cushions that may supply mobility between the collagen fibrils as

introduced in previous studies [31, 24].

Limitations

Although providing additional insight into tendon mechanics, these studies do have

some limitations. The optical measurement of soft tissue strains proposed in Chap-

ter 3 improved the analytical precision in examining the substructures; however

these measurements were limited by the assumption that surface strains are indica-

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Synthesis

tive of the strains inside the various tendon subregions. These measurements were

not sufficiently sensitive to detect fiber uncrimping [47] or to determine whether

inter-fascicle slippage occurred [48, 49]. Finally, the complex hierarchical collagen

structures of tendon do not generally represent the response of a continuum ma-

terial. Despite these limitations, useful insight into local tendon mechanics could

be extracted; the softening of the bone-tendon-muscle complex was identified as an

alteration of the bone insertion, solely, rather than in all three tissues.

For the analysis of the fibril morphology presented in chapter 4.1, a limitation that

one could argue is that the sample preparation protocol does not allow morphological

analysis in the same tendon that was mechanically tested to failure. In this study it

was critical to analyze the morphology of the tendon structure in the tensile region

which would not have been possible after rupture of the tendon. Despite this,

the results revealed a significant trend that relates PG and collagen morphometry

together with tendon mechanical behavior in tension.

The two AFM studies presented also have some limitations. First, the amount

of creep experienced during chemical fixation of the tissue with glutaraldehyde [23]

was relatively uncontrollable (although generally consistent from sample to sample).

However, the majority of the creep deformation occurs within the first minute and

is limited afterward, and further, in relaxation experiments it was shown that the

largest effects at the fiber and fibril levels occur within 20 s of the applied load

[2]. Creep loading and uncontrolled dynamics of chemical fixation could therefore

affect the resultant fibril structure, although whether and how this may influence the

results is difficult to predict. While we made an effort to minimize such effects by

mechanical preconditioning, effects of and differences between creep and relaxation

remain ground for future work. As a second limitation, the preparation of high

quality slices for AFM analysis was experimentally challenging. Before sectioning,

all samples were embedded in a medium containing polyvinyl alcohol which is known

to form thin films and can leave surface residues. While we have confidence in

the fidelity of the image data we did not exclude the possibility that film residues

may have adversely affected morphometric accuracy of the subsequent d-period and

diameter measures.

Conclusion and future work

In conclusion, tendon is a complex material that can not be modeled as a simple

composite material. Fibril density is not directly related to tendon stiffness and the

PGs do not appear to transfer load between the collagen fibrils. GAG concentration

does not significantly influence the overall tendon mechanical response, most likely

106

References

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are responsible for changes in collagen fibril elongation, which increases at lower

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“mechanical role” of GAG does not appear to be linking the collagen fibrils in order

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In conjunction with previous studies [2, 21, 45], the measured fibril elongation

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tions should be performed to understand whether there is lost energy, or if is it a

consequence of the fibril sliding.

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113

Abbreviations

Abbreviations

2D two-dimensional

3D three-dimensional

AFM Atomic force microscope

BEC BEST Engineering Cluster

BEST BioEngineering, BioSystems and BioTechnology

B6 C57BL/6 genomic group

CB cupromeronic blue dye

CS chondroitin sulfate

C3H C3H/HeJ genomic group

DMMB dimethylmethylene blue

DS dermatan sulfate

ECM extracellular matrix

EDTA ethylenediaminetetraacetic acid

E-modulus elastic modulus

EPFL Ecole Polytechnique Federale Lausanne

ETH Eidgenossische Technische Hochschule

EU European Union

GAG glycosaminoglycan

GDF Growth/Differentiation Factor

HA hyaluronic acid

KS keratan sulfate

KTH kungst tekniska hogskola

µCT microcomputed tomography

MSC mesenchymal stem cell

PBS phosphate buffered saline

PEG polyethyleneglycol

PG proteoglycan

ROI region of interest

SD standard deviation

SEM scanning electron microscopy

TEM transmission electron microscopy

114

Abbreviations

115

List of Figures

2.1 Function related to the structure of the same ground material. The

carbon atom with a different atomic structure is the ground material

of two totally different materials like diamond and graphite. . . . . . 9

2.2 Schematic section of a rope. . . . . . . . . . . . . . . . . . . . . . . . 10

2.3 Typical stress-strain curve and corresponding images at the

macroscale (visualized using polarized light) and microscale (struc-

tural changes occurring at the fibrillar level) of a rat tail fascicle. In

the toe region the macroscopic crimps disappear, in the heel region

the molecular kinks in the gaps regions progressively straighten, while

in the elastic region the intermolecular spaces elongate and the fibril

order increases. Figure adapted from Fratzl [45]. . . . . . . . . . . . . 12

2.4 Organization of tendon as identified from nanoscale imaging tech-

niques (x-ray diffraction, electron microscopy) to microscale (scanning

electron microscopy, visible light microscopy). This classic figure is

adapted from Kastelic [1]. . . . . . . . . . . . . . . . . . . . . . . . . 13

2.5 Schema and micrographs of the d-period. The schema on the left

shows the d-period regular stripe visible on the collagen fibrils sur-

face along the longitudinal axis. On the right side, experimental view

of the d-period with different imaging techniques: atomic force micro-

scope (AFM), scanning electron microscopy (SEM) and transmission

electron microscopy (TEM). . . . . . . . . . . . . . . . . . . . . . . . 14

2.6 Formation and self-assembly of collagen fibrils. Procollagen is se-

creted from the cell and cleaved at the propeptide ends to form a

collagen molecule. The fibril is formed by the spontaneous parallel

self-assembly of the collagen molecules. As a last step the fibrils are

stabilized by covalent cross-linking, which is initiated by lysil oxidase. 15

117

List of Figures

2.7 Schematic representation of a tendon fibril. The spontaneous lateral

arrangement of the collagen molecules results in an overlap of the

molecules (labeled with O). The gap region (labeled with G) appears

in one molecule out of five and results in the d-period. Figure is

adapted from Hodge-Petruska [23]. . . . . . . . . . . . . . . . . . . . 16

2.8 Schema of a proteoglycan aggrecan and decorin. The aggrecan is

a large protein, to which several glycosaminoglycans are covalently

connected to the core protein, while the decorin is the smallest PG

and has only one GAG chain. Adapted from Alberts et al [32]. . . . . 17

2.9 Cross-sectional histological slide of (left) healthy control mouse

Achilles tendon, and (right) healing Achilles tendon at 42 days post-

operation. The bar indicates approximately 125 nm [12]. . . . . . . . 18

2.10 Schema of the similar function in tension of a tendon and a com-

posite material. The tendon structure is composed of collagen fibrils

connected with PGs within the ECM and the composite material is

composed of a stiff fiber embedded in a softer matrix that follow the

shear-lag theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.1 Typical stress-strain curve for an Achilles tendon during uniaxial ten-

sile testing (left). Optical strains were extracted from video frames

corresponding to points A and B indicated in the linear region of the

test curve. A is the end of the toe region, defined at 0.8 N; B cor-

responds to 80% of the ultimate load. Displacement markers (high-

lighted here for emphasis) were automatically detected according to

regions of high image contrast. . . . . . . . . . . . . . . . . . . . . . . 35

3.2 Posterior view of a murine Achilles tendon clamped for tensile test-

ing. The optical strain was calculated using video of the visible tendon

fibers, while machine-based strain was automatically determined from

the machine clamp to clamp distance. For a focused local strain anal-

ysis, the tendon midsubstance was evenly divided into three parts,

the proximal region included a portion of the tendon to the intra-

muscular fibers, the central region comprised the middle third of the

tendon midsubstance, and the distal region included the transition

from midsubstance to the tendon-bone insertion. . . . . . . . . . . . . 36

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3.3 Chondroitin sulfate weight per wet tendon weight (µm/mg) for the

three midsubstance regions as determined by GAG assay. The relative

subregion contribution to total midsubstance GAG content is also

shown as a percentage. All columns connected by a star (?) are

statistically different at p < 0.05. . . . . . . . . . . . . . . . . . . . . 37

3.4 (A) Median stress vs strain curves for control and GAG digested

murine Achilles tendon. After digestion, the GAG-depleted group

(dashed gray) shows a lower modulus than the control group

(black). (B) The regression line indicates the relationship between the

machine-based elastic modulus and the optically-based elastic modu-

lus for both the control and the digested groups. . . . . . . . . . . . . 39

3.5 Box plot of (A) the strain and (B) the elastic modulus in loaded

tendon, as measured in the proximal, central and distal region of the

tendon midsubstance. The distal subregion of GAG-depleted tendon

was statistically different from corresponding controls with p-value <

0.05. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4.1 Representative transmission electron micrograph for the two inbred

strain; B6 and C3H, and its binarized reconstruction on the right.

The binarized reconstruction is processed to calculate the fibril ra-

dius, the collagen area fraction, the interfibrillar distance, the specific

fibril surface and the fibril contact area. The color bar indicates the

pixel size of the fibril radius that is converted to nm. The black bar

indicates 400 nm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

4.2 Schematic representation of the morphological parameters measured

with the automatic script. (A) Fibril radius, (B) collagen area frac-

tion, (C) interfibrillar distance, (D) specific fibril surface, (E) fibril

contact area. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4.3 Cumulative distribution curves for B6 and C3H mice. B6 generally

contain a majority of small fibrils, while C3H have a more uniformly

distributed fibril radius. Histograms of the collagen fibril distribution

for both groups. n is the total number of fibrils. . . . . . . . . . . . . 56

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4.4 Representative transmission electron micrograph of a longitudinal

section of the Achilles tendon for (A) B6 and (B) C3H at a magnifi-

cation of ×53000. The black lines between the fibrils are the stained

GAGs. (C) Means and standard deviations of sulfated GAG content

in the two groups. * statistically significant for p-value < 0.05. . . . . 57

4.5 Experimental setup for fixing the strained tendon at a given load. The

tendon is clamped to a universal tensile test machine in a chamber

that is filled with 1.25% glutaraldehyde solution, while the tendon is

under constant load for 8 h. The applied constant load (input) and

the tendon strain response (output) are recorded. The fixed tendon

is sliced and the fibril strain is measured with an AFM. The images

show the d-period observed with AFM, scanning electron microscopy

(SEM), and transmission electron microscopy (TEM). . . . . . . . . . 71

4.6 (A) AFM height image of collagen fibrils taken in contact mode, (B)

two-dimensional profile of the z-axis corresponding to the line on im-

age A. (C) Detail of a typical power spectrum density of the 2-D

profile; the highest peak in the graph corresponds to most frequent pe-

riodicity in the two-dimensional profile of B. The standard dimension

of the x-axis is 1/nm, in this case the inverted scale is also displayed

below, and the peak corresponds to 66.4 nm. . . . . . . . . . . . . . . 72

4.7 (A) AFM height image of collagen fibrils taken in contact mode, (B)

the two-dimensional profile of the dash line, and (C) schematic in-

terpretation of fibril arrangement from the profile. Some fibrils can

be easily recognized on the profile (e,f,g,h), while others have a more

complicated arrangement and do not show a clear round profile. In

(C) the 2D profile from inset (B) is displayed to show the correspon-

dence with the collagen fibril diameter. On the fibrils the AFM trace

is drawn to scale, while in the line above the trace is amplified 33

times. The pink semi-circles represent the tip in contact to the surface. 73

4.8 Probability distribution of the collagen fibril d-period length as a

function of the measured strain on the tendon. The d-period length

is increasing with increasing total strain of the tendon, whereas the

variability of the d-period length is decreasing with increasing strain. 76

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List of Figures

4.9 Height AFM images of collagen fibrils taken in contact mode for (A)

an unloaded tendon, and (B) a tendon stretched to 10% strain. The

z-range is indicated by grayscale bar with values in nm. . . . . . . . . 76

4.10 Plot of the applied tension for the bulk tendon, the measured d-period

length of the collagen fibrils and the mean collagen fibril diameter

as a function of the target strain. At 10% strain, the increase in

the d-period length and the decrease in fibril diameter is statistically

significant (* indicates a p-value < 0.05). No significant difference

was found between the 0% and 5% strain groups. . . . . . . . . . . . 77

4.11 Schema of the mechanical behavior of collagen fibrils connected by PG

cross-links. (top) Fibrils in a high cross-linked network slide and elon-

gate homogeneously to bear the load. (bottom) For GAG-depleted

tendons, we hypothesize that the fibrils are loosely coupled showing

a lower fibril strain and a higher inter-fibril gliding than a native ten-

don. If the fibrils are tightly coupled, the total fibril strain would

increase and the the inter-fibril gliding decreases. . . . . . . . . . . . 88

4.12 Experimental set-up of the tendon preparation and analysis. A) The

tendon is clamped in a tensile test machine under constant load to

reach a defined target strain. The chamber is filled with 1.25% glu-

taraldehyde to fix the tendon chemically while submitted to constant

load. B) Representative AFM height image of a tendon fascicle show-

ing collagen fibrils characterized by the repeated gaps forming the

d-period. C) Two dimensional height profile of the black line in the

image B, and D) result graph obtained after Fourier function trans-

form analysis of the two-dimensional profile from Figure 4.12c.The

highest peak corresponds to the inverse of the length of the most

frequent periodicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

4.13 Representative transmission electron micrograph (A,C) and AFM

height images (B,D) of the longitudinal section of a native control

(A, B), and a dehydrated Achilles tendon (C, D). The plot displays

the mean d-period length and collagen diameter measured for native

and digested tendons that were normally incubated in PBS (control

group), or successively dehydrated with PEG (dehydrated group). . . 94

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List of Figures

4.14 Mean fibril diameter and d-period length versus total measured target

strain in the tendon for native and digested tendons. In the native

tendon the d-period length is statistically longer for 15% in compar-

ison to the 5% strain group. The digested tendon shows a regular

increase in the d-period length that is significant at 15% strain with

both the 0% and 5% target strain group. At 15% strain a statis-

tical difference is showed between the native and digested tendons.

For both native and digested tendon group the diameter is gradually

decreasing and is statistically smaller over 5%. . . . . . . . . . . . . . 95

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List of Tables

2.1 Collagen types present in the body. . . . . . . . . . . . . . . . . . . . 14

3.1 Summary of the measured parameters. . . . . . . . . . . . . . . . . . 38

3.2 Elastic modulus values for the tendon midsubstance and its three

component subregions, based on local strain measurements. . . . . . . 38

4.1 Summary of measured morphological parameters measured for B6

and C3H mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.2 Summary of the measured mechanical parameters for B6 and C3H

mice. The E-modulus is showed as a median (first quartile; third

quartile). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

4.3 Relative homogeneity of collagen fibril loading in tendon (indicated by

% of normally distributed measures). While some heterogeneity was

observed in isolated regions of analysis in loaded tendons, collagen

fibrils strains were generally homogeneous. . . . . . . . . . . . . . . . 74

4.4 Summary of fibril structure characterization. A,B,C,D statistical sig-

nificant for p-value < 0.05 . . . . . . . . . . . . . . . . . . . . . . . . 75

4.5 Incubation effect on Type I collagen’s d-period and fibril diameter at

0% strain. The control group was incubated in PBS and successively

chemically fixed, the dehydrated group was incubated in PBS, in

PEG and successively chemically fixed and the digested group was

incubated in PBS with ChABC and chemically fixed. . . . . . . . . . 93

123