TEL AVIV UNIVERSITY The George S. Wise Faculty of Life Sciences

Department of Zoology

Biological, biochemical, and mechanical properties of

collagen fibers

of the soft coral Sarcophyton ehrenbergi

THESIS SUBMITTED FOR THE DEGREE "DOCTOR OF PHILOSOPHY" BY

IDO SELLA

SUBMITTED TO THE SENATE OF TEL AVIV UNIVERSITY March 2012

This work was carried out under the supervision of

Prof. Yehuda Benayahu

Acknowledgments

I would like to express my gratitude to many people who helped me during the “fiber’s quest”.

To Prof. Yoal Kashman and Dr. Amira Rudi who guided me in the mysterious world of biochemistry.

To Prof. John Gosline, Prof. Yoram Lanir and the good people of Friday Harbor marine station who introduced me to beauty of biomechanics.

To Prof. Joseph P. R. O. Orgel, and Prof. Felix Frolow for valuable assistance, and willingness to share their enormous knowledge in X-ray diffraction.

To Dr. Yaniv Assaf for valuable assistance in MRI.

To Yakov Delaria for his valuable work in Electron Microscopy and some great cups of coffee.

To Naomi Paz for editorial assistance, Moshe Aleksandroni for his help with photography, and Varda Wexler for graphic assistance.

To Mati Halperin and Chen Yoffa for the endless support and friendship

To Shimrit Perkol-finkel for pushing me forward in the last 9 years

To my lab mates who helped me above and underwater and Nissim Sharon in particular.

To the staff of the Interuniversity Institute of Marine Biology at Eilat for the kind hospitality.

To my parents Avner and Varda, my sisters Ronit and Michal, and to Emma my lovely, optimistic and beautiful wife, for supporting me along this long way.

Table of Contents Acknowledgments

Table of contents

Figure list

Table list

Formula list

Abstract ....................................................................................................................... i

1.Introduction .............................................................................................................1

1.1 Structural features of Octocorallia .....................................................................1

1.2 Features of the mesoglea ................................................................................5

1.3 Properties of collagen .......................................................................................8

1.4 Evolution and characteristics of collagen among invertebrates ....................... 13

1.5 Objectives ....................................................................................................... 17

2. Materials and Methods ......................................................................................... 20

2.1 Collection of colonies ...................................................................................... 20

2.2 Farming of colonies ......................................................................................... 20

2.3 Extraction of fibers .......................................................................................... 21

2.4 Proton and carbon nuclear magnetic resonance analyses (NMR) ................... 21

2.5 Amino acid analysis ........................................................................................ 22

2.6 Light and electron microscopy ........................................................................ 23

2.7 Graphic analysis ............................................................................................. 24

2.8 Wide-angle X-ray diffraction ............................................................................ 25

2.9 Magnetic resonance imaging .......................................................................... 26

2.10 Biomechanical studies .................................................................................. 26

2.11 Thermogravimetric analysis and differential scanning calorimetry ................ 30

3. Results ................................................................................................................. 31

3.1 Biochemical and structural properties of fibers ................................................ 31

3.1.1 Proton and carbon nuclear magnetic resonance .............................................. 31

3.1.2 Amino acid analysis ......................................................................................... 32

3.1.3 Light and electron microscopy ......................................................................... 35

3.1.4 Wide-angle X-ray diffraction ............................................................................ 42

3.2 Location, distribution and formation of fibers within colony .............................. 43

3.2.1 Light and electron microscopy ......................................................................... 43

3.2.2 Magnetic resonance imaging ........................................................................... 49

3.3 Biomechanical and physical properties of fibers .............................................. 50

3.3.1 Biomechanical properties of isolated fibers ...................................................... 50

3.3.2 Mechanical characterization of fibrils SPM-TEM in situ study .......................... 52

3.3.3 Thermogravimetric analysis ............................................................................. 55

3.3.4 Differential scanning calorimetry ...................................................................... 55

4. Discussion ............................................................................................................ 58

4.1 Proton and carbon nuclear magnetic resonance and amino acid analyses ..... 58

4.2 Light and electron microscopy ........................................................................ 60

4.3 Distribution and formation of fibers .................................................................. 65

4.4 Biomechanical and physical properties of fibers .............................................. 70

4.5 Summary ........................................................................................................ 74

5. References ........................................................................................................... 76

Hebrew abstract Figure List Fig. 1. Collagen molecule .........................................................................................9

Fig. 2. Collagen structure ........................................................................................ 12

Fig. 3. Biomechanical experimental set-up .............................................................. 27

Fig. 4. Isolated fibers ............................................................................................... 31

Fig. 5. NMR spectroscopic profile of fibers ............................................................... 33

Fig. 6. Amino acid analysis of fibers ......................................................................... 34

Fig. 7. Histological sections of fiber-bundles ............................................................ 37

Fig. 8. TEM of fibers ................................................................................................. 39

Fig. 9. TEM of fibrils and graphical analysis ............................................................. 40

Fig. 10. TEM of longitudinal sectioned isolated fiber ................................................. 41

Fig. 11. Wide-angle X-ray diffraction of fibers ........................................................... 42

Fig. 12. Light microscopy of fibers within polyp ......................................................... 45

Fig. 13. TEM of gastrodermal cells within mesentery ................................................ 46

Fig. 14. Microscopy of mesoglea .............................................................................. 47

Fig. 15. Light microscopy of colony’s stalk ................................................................ 48

Fig. 16. Magnetic resonance of colony ..................................................................... 49

Fig. 17. Mechanical properties of isolated fibers ....................................................... 51

Fig. 18. Mechanical properties of in vivo fibril ........................................................... 53

Fig. 19. In situ mechanical characterization of fibril by SPM-TEM............................. 54

Fig. 20. Thermogravimetric analysis of isolated fibers .............................................. 56

Fig. 21. Differential scanning calorimetry of isolated fibers ....................................... 57

Table List Table 1. Amino acid characterization of fibers of Sarcophyton ehrenbergi ................ 35

Table 2. The soft coral. Summarize of material properties of fibers of Sarcophyton

ehrenbergi in comparison to other known collagens ................................................. 71

Formula List Biomechanical study of the fibers: transformation of the results to Strain (e) and

Stress (s) values: s=F/A e= ΔL/L0 ........................................................................... 28

i

Abstract

Cnidarians are polypoid or medusoid organisms that feature a radial or biradial symmetry.

Their body encloses a cavity with a single opening surrounded by tentacles. They are

diploblastic, consisting of two cell layers, the endoderm and the ectoderm, which are

separated by the a-cellular matrix, termed the mesoglea. The class Anthozoa (phylum:

Cnidaria) comprises mainly benthic life forms and characterized by two anatomically-related

structures, a tubular gullet (pharynx), and the mesenteries which constitute sheets of tissue

that extend in a radial manner from the body wall to the pharynx. The subclass Octocorallia (

class: Anthozoa) includes an estimated 3,200 species from the orders Alcyonacea,

Pennatulacea, and Helioporacea. Octocorallia are colonial with a “colonial tissue”, termed

coenenchyme, located between the polyps, consisting mostly of the mesoglea and sclerites.

Within the coenenchyme there is a network of solenia and wider gastrodermal canals. The

polyps feature eight complete mesenteries and possess eight tentacles, each bearing two

lateral rows of feather-like pinnules. The mesoglea occupies a relatively large portion of the

biomass of octocoral colonies, and possesses features that are remarkably similar to the

extracellular matrix of other metazoans. The role of the mesoglea is primarily a mechanical

one, as it provides structural reinforcement and stiffness to maintain the hydrostatic skeleton

and reinforce muscle-action. It is a pliant composite material with a highly hydrated matrix of

polysaccharides and proteoglycans, encompassing discontinuous collagen fibers. Collagen

is one of the most abundant proteins in animals. It is arranged hierarchically, and can

present different structures, ranging from gelatinous to fibrous rope-like structures. All

collagens are composed of three polypeptide chains that are wound around one another to

form superhelix tropocollagen molecules. These molecules form collagen microfibrils/ fibrilis

which, in turn, can form collagen fibers. The types of collagen differ from each other in the

ii

composition of the amino acids in the polypeptide chains and in the composition of chains

within the tropocollagen molecule.

The overall objective of the present study was to identify and characterize the fibers found in

colonies of the reef-dwelling zooxanthellate octocoral, Sarcophyton ehrenbergi (family

Alcyoniidae) in order to define a new morphological finding, toevaluate their possible

practical application, and to view there rule in the evolution of collagen. The fibers are

located within the colony and, when mechanically extracted, they feature bundles that may

reach hundreds of microns in diameter and up to tens of centimeters in length. Nuclear

magnetic resonance analyses revealed that the fibers are composed of protein. Amino acid

analyses supported the hypothesis that the fibers are collagenous, in having a high

concentration of glycine, proline and hydroxyproline. The study deals with the biochemical,

structural, biomechanical, and physical properties of the fibers, including their location at the

ultrastructural level. The study applied microscopy, nuclear magnetic resonance analyses,

biochemical and material analyses, such as thermogravimetric analysis and differential

scanning calorimetry, and biomechanical techniques. Collagen-specific histological staining

of isolated bundles of fibers confirmed their collagenous nature, and revealed a packed

arrangement of almost round fibers in cross-section, coiled around each other. The diameter

of the fibers (9 ± 0.37 µm, n=166 fibers) is significantly smaller than that of collagen fibers

found in connective tissues of vertebrates (50-300 μm). The fibers and fibrils of the soft coral

are organized similarly to collagen types I - III. The fibrils also revealed thinner sub-units

(~2.5 nm wide) tightly packed, which are assumed to be an external projection of the micro-

fibrils or collagen molecules. Cupromeronic-Blue staining revealed a densely hydrated

matrix of PGs in the soft coral collagen. This was also observed in wide-angle X-ray

diffraction, which presented a water-rich fibrilar structure. Additionally, this latter method

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confirmed the fibrilar nature of the soft coral collagen, its fibrilar packing function, D-

periodicity, helix pitch, and helix molecular packing. Although resembling the arrangement

of tendons and ligaments, the water-retentive capacity of the fibers was more structurally

related to the mesoglea. This may indicate some kind of intermediate state between the

gelatinous mesoglea and fibrous tendons and ligaments, as the ability to strongly retain

water is one of the properties that defines the differences between these two collagen

structures. Thermogravimetric analysis (TGA) provided additional evidence for the water

retentive capacity of the fibers, as stored and dried fibers featured 7-30% weight loss upon

heating to ~1000C. Differential scanning calorimetry revealed that the soft coral collagen

exhibits an unexpectedly high denaturation temperature of 67.8°C, similar to artificially

cross-linked collagen. Biomechanical study of the fibers revealed a set of properties

(stretching ability 19.4±4.27%, n=12; stiffness 0.44 ± 0.1 GPa, n =12; and average stress to

failure 49.4 ± 11.7 MPa, n=12) that are more closely related to tendons than mesoglea.

Imaging of a whole colony and microscopy of its different parts identified the location and

distribution of the fibers. Packed coiled collagen fibers lay along six out of the eight

mesenteries of the gastrovascular cavity of the polyps. They extended from the polyps to the

stalk via mesentery-like-structures of the gastrodermal cavities, into the basal part of the

colony. The location of these fibers and their biomechanical properties may be an indication

of their functional role, in providing structural support for both the polyps and the whole

colony. The study also revealed laminated collagenous fibers within the mesoglea around

the sites of decalcified sclerites. These laminated fibers differed from the mesenterial ones,

although both were associated with striated vesicles located within the surrounding cells.

The location, size (>500 nm,) and the striated appearance of these vesicles may suggest

their possible role in fibrillogenesis.

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To date, among invertebrates, there is no record of any internal long collagenous fibers, like

those of S. ehrenbergi that can be observed by the naked eye. The finding of tendon-like,

long collagen fibers in a two cell-layered organism, may change our perspective on the

appearance, diversity, and development of collagens in metazoans. The fibers of S.

ehrenbergi present a mixed set of properties where some resemble those of vertebrates,

some of invertebrates, and others are novel. A better understanding of the molecular

structure and a genomic study of cells containing the striated vesicles, may contribute to the

understanding of collagen evolution and fibrillogenesis. Moreover, further elucidation of the

features of this collagen will enable examining the feasibility of using the fibers for

biomedical applications. S. ehrenbergi collagen fibers present a set of properties such as

thermal stability, fibril organization, biomechanical and water-retentive capacities that are

suitable for tissue engineering and medical devices. Certain preliminary findings also

indicate that the fibers may contribute to taxonomic identification of certain alcyoniid

octocorals. The current study findings from both field and laboratory studies, consequent

results of both scientific and practical importance.

v

1

1. INTRODUCTION 1.1 Structural features of Octocorallia

Cnidarians are polypoid or medusoid organisms that feature a radial or

biradial symmetry (Fautin and Mariscal, 1991). They are diploblastic,

consisting of two cell layers, the endoderm and the ectoderm. These are

separated by a-cellular matrix, called the mesoglea (Ruppert et al., 2004).

The cnidarian body encloses a cavity, termed the coelenteron or

gastrovascular cavity, which opens to the environment through a single

opening and is surrounded by tentacles. This cavity serves for gas exchange,

food digestion, and reproduction (Pechenik, 2000). The tentacles possess

stinging cells (nematocytes) used for predation, offense and defense (Fautin

and Mariscal, 1991). Both the polyp and medusa forms, as well as the

nematocysts, are unique characteristics of members of the phylum Cnidaria

(Daly et al., 2007).

Most cnidarians are carnivores, while some are herbivores (Fabricius, 1995),

and they are generally regarded as passive predators that use their

nematocytes when feeding on the prey items that pass through their tentacles

(Fautin and Mariscal, 1991). In addition to their predator abilities, numerous

species are associated with symbiotic unicellular algae (zooxanthellae), and

thus enjoy a constant supply of photosynthates (Van Oppen et al., 2005).

There are also some cnidarians that can absorb dissolved organic matter

from the seawater (Schlichter, 1982). Regardless of whether they possess a

mineral or an organic supporting structure, all cnidarians feature a hydrostatic

skeleton, in which the muscles of the body wall operate against the fluid of the

2

gastrovascular cavity in order to expand the polyps, or to generate movement

(Koehl, 1984). These features contribute to the ability of cnidarians to occupy

a variety of habitats along the water column from the tropics to the poles, and

thus to exhibit a worldwide distribution, mainly in the marine environment but

also in fresh water (Pechenik, 2000).

The phylum Cnidaria is classified into four classes: Anthozoa

Scyphozoa, Cubozoa, and Hydrozoa (Fautin and Romano, 2000). Following a

certain pelagic phase the cnidarian planulae-larvae settle and attach to the

substrate, where they metamorphose into a polyp-stage, which will

subsequently mostly bud off additional polyps to form a colony (Ruppert et al.,

2004). Among anthozoans, the polyps or the colony become sexually

reproductive, whereas in other cnidarian classes the medusae possess this

function (Daly et al., 2007). The class Anthozoa is composed mainly of

benthic life-forms (Pechenik, 2000). Morphologically, anthozoans are

characterized by two anatomically-related structures, the actinopharynx and

the mesenteries, which are unique to cnidarian polyps (Fautin and Romano,

2000). The actinopharynx is a tubular gullet, extending from the mouth

opening into the coelenterons, and containing at least one flagellated

longitudinal channel, termed the siphonoglyph (Ruppert et al., 2004). In most

sea anemones and corals, two siphonoglyphs are situated diametrically

opposite one another in the actinopharynx, and propel water in and out of the

coelenterons (Pechenik, 2000). The mesenteries are longitudinal sheets of

tissue that extend in a radial manner from the body wall; some (complete/

perfect) reach all the way to the actinopharynx, while others have a free edge

3

which is suspended within the coelenteron (Fautin and Romano, 2000). The

free edge of a mesentery may feature filaments provided with cilia, gland

cells, and cnidae. The cilia probably circulate fluid that otherwise might

stagnate in the compartments defined by the mesenteries. The cnidae and

gland cells function in digestion (Fautin and Mariscal, 1991). It is inferred that

the mesenteries increase the surface area for respiration and absorption of

food, and provide mechanical support for the body wall (Fautin and Romano,

2000). The longitudinal retractor muscles of the mesenteries enable the

polyps to retract. The gametogenic tissue of anthozoans lies between these

muscles and the edge of the mesenteries (Ruppert et al., 2004). Some

colonial anthozoan species display polymorphism, having polyps specialized

for functions such as feeding or water circulation (Fautin and Mariscal, 1991).

The class Anthozoa is divided into the subclasses Hexacorallia and

Octocorallia. Hexacorallia, among others, include the orders Corallimorpharia

(jewel anemones), Actiniaria (sea anemones), Zoantharia (encrusting

anemones), and Antipatharia (black corals) (Berntson et al., 1999; Chen et al.,

1995; Daly et al., 2007). Octocorallia include the orders Alcyonacea (soft

corals, sea fans and sea whips), Pennatulacea (sea pens), and Helioporacea

(blue corals) (Fabricius and Alderslade, 2001). Octocorallia are colonial with a

“colonial tissue”, termed the coenenchyme, situated between the polyps,

consisting mostly of mesogloea and sclerites, and penetrated by a network of

solenia and larger gastrodermal canals (Bayer et al., 1983). Octocoral polyps

possess eight tentacles, each bearing two lateral rows of feather-like pinnules

(Fabricius and Alderslade, 2001). Additionally, they feature eight complete

mesenteries, so that the gastrovascular cavity between a pair of mesenteries

4

extends to each tentacle (Fautin and Romano, 2000). The actinopharynx of

each polyp has a single siphonoglyph, and most Octocorallia has both an

hydrostatic skeleton and an endoskeleton (Fautin and Mariscal, 1991). The

latter is either in the form of calcareous sclerites, embedded in the mesoglea,

or an internal rod-like axis of calcareous or organic-horny substance, as found

in sea fans and sea whips (Chang et al., 2007).

Octocorals are known from all the worlds’ oceans and at all depths (Devictor

and Morton, 2010). There are approximately 340 genera of octocorals from 46

valid families, with an estimated number of 3,200 species (Devictor and

Morton, 2010; Williams, 1995). Their world-wide dispersal and confused

taxonomy (McFadden et al., 2006), have made octocorallia a subject of recent

molecular phylogenetic studies (Daly et al., 2007; McFadden et al., 2011).

The confused state of taxonomic relationships within and between some

genera of octocorals, is a combined product of the relatively few

morphological characters available, lack of understanding of intraspecific

variation in those morphological characters, and historical lack of taxonomic

work (McFadden et al., 2006). The form and distribution of sclerites

embedded in the coenenchymal tissue and polyps, and the overall colony

growth, are the most important characters used to distinguish genera and

species of octocorals (Daly et al., 2007). The use of molecular markers to

examine the phylogenetic and taxonomic relationships among octocoral

species has contributed significantly to the ability to differentiate species. This

is most important in cases in which the morphological identification of genera

is blurred, such as intermediate colony growth form, or disparity between

colony growth form and the form or distribution of sclerites in its tissue

5

(McFadden et al., 2006). It should be noted that taxonomic identification is

becoming increasingly important in light of the diverse content of natural

products found in octocorals, which has made them a target for biochemical

and biomedical oriented research (Blunt et al., 2009; Look et al., 1986;

Nguyen Xuan Cuong, 2008; Tanaka et al., 2005).

1.2 Features of the mesoglea

The relative volume and quantity of the mesoglea differ among the four

cnidarian classes (Fautin and Mariscal, 1991). The Scyphomedusae have an

a-cellular mesoglea that constitutes the vast bulk of the animal, while in the

hydroids it comprises little more than a-cellular glue, holding the cell layers

together. Anthozoans possess intermediate dimensions of mesoglea: the

Hexacorallia feature a relatively small quantity while in the Octocorallia it

occupies a larger portion of the colony biomass (Pechenik, 2000).

Ultrastructural and biochemical studies of the mesoglea, and genomic

analysis of cells within it, have revealed features that are remarkably similar to

the extracellular matrix (ECM) of other metazoans (Tucker et al., 2011). For

example, in Hydra vulgaris (Pallas,1766) and in some hydrozoan medusae,

the mesoglea is organized in the morphological form of a basal lamina,

composed of a meshwork of thin filaments underlying the epithelial cell layer

(Davis, 1975). Clusters of cells within the mesoglea of H. vulgaris revealed the

presence of genes originating from the common ancestral ones related to

collagen type IV (see below) and laminin (Fowler et al., 2000; Shimizu et al.,

2009; Zhang et al., 2002), both of which are found in most metazoan ECM

(Har-el and Tanzer, 1993).

6

As in vertebrate ECM, the role of the mesoglea is primarily a mechanical one,

being crucial in providing structural reinforcement, controlling shape,

transmitting stresses, storing elastic energy, and providing stiffness to

maintain hydrostatic skeleton and reinforce muscle action (Alexander, 1962;

Chapman, 1953; Elder, 1973; Gosline, 1971; Koehl, 1977; Thompson and

Kier, 2001). The mesoglea is a pliant composite material with a discontinuous

fiber system within a highly hydrated matrix (Koehl, 1977). The mesogleal

matrix is a diluted network of randomly-coiled polymers, such as

polysaccharides and glycoproteins that are lightly cross-linked to each other

by electrostatic and covalent bonds, and the fibers comprised of collagen

fibrils or fibers (see below) (Chapman, 1953; Gosline, 1971; Vogel, 2003).

The mesoglea behaves in a viscous-elastic manner, which is consistent with

its biochemical composition. Chapman (1953) demonstrated the pliable

properties of the mesoglea when strips removed from the sea anemone

Metridium senile were sectioned in different directions and revealed little

difference in tensile strength. Alexander (1962) found that the mesoglea of M.

senile and another sea anemone, Calliactis parasitica, could be stretched to

three times its original length by a small stress (~2x104 dynes/cm2), applied

for periods of 10-20 hours. It would then nearly return to its original un-

stretched length over the same time period following removal of the stress.

This extensibility and elasticity of the mesoglea is suggested to be due to the

random-coil polymers in its matrix, rather than to its collagen fibers (Vogel,

2003). As the mesoglea is stretched, the collagen fibers tend to align with the

stress axis, slide past each other, and deform the matrix molecules between

them (Gosline, 1971; Wainwright et al., 1976). The closer the collagen fibers

7

are to each other (relative concentration in the matrix), and the closer they are

to being parallel with the stress axis, the more rigid the whole structure will be

(Vogel, 2003). These are the main properties that distinguish the gelatinous

mesoglea from the rigid convective tissues, such as tendons and ligaments,

found in vertebrates (Alexander, 1962; Fautin and Mariscal, 1991; Gosline et

al., 2002). Both the mesoglea and tendons are composed of fiber and matrix,

but in the latter the fibers are arranged in parallel with much less matrix,

causing the tendon to behave mechanically as a continuous composite

material (Vogel, 2003).

Although the mesoglea’s role is primarily a mechanical one, it also has other

functions (Fautin and Mariscal, 1991). For example, in Octocorallia, in addition

to acting as a reinforcement layer, where calcareous sclerites are embedded

within the amorphous matrix, the mesoglea contains clusters of cells in a

similar way to those in connective tissue (Fautin and Mariscal, 1991; Koehl,

1982). The most typical cells found there are irregularly-shaped amoebocytes

and scleroblasts, which are derived from the ectoderm (Fautin and Mariscal,

1991). The former are thought to be involved in the production of collagen

fibrils and in the movement of food as well as waste material (Larkman, 1984),

and the later produce the sclerites (Jeng et al., 2011; Meszaros and Bigger,

1999). Meszaros and Bigger (1999) demonstrated how the mesoglea

functions in wound healing of the octocoral Plexaurella fusifera, as

regeneration seems to be related to the amoebocytes that extrude the

connective fibers necessary for this process. The mesoglea is also involved in

the provision of a base upon which the muscle fibers are mounted, and allows

their change in length. Batham and Pantin (1951) revealed that regions of the

8

mesoglea which accommodate muscle fibers are much less extensible or

deformable than regions of the mesoglea lacking these fibers, and that the

latter accommodate the buckling of the former when muscular contraction

takes place. Another interesting function of the mesoglea is its ability to store

different substances (Fautin and Mariscal, 1991). It serves as an additional

fluid reservoir antagonist to muscles, and takes part in the temporary storage

of chemical energy and essential minerals. Hamner and Jenssen (1974)

found that in the scyphomedusa Aurelia aurita, the size of the mesoglea

rapidly diminishes upon starvation, and that the animal becomes shrunken

and distorted within a number of weeks, in the same way that starvation

influences the reduction and utilization of fat storages in vertebrate bodies

with time.

Being an a-cellular layer, most of the biological properties associated with the

mesoglea result from its composition and structure. The amount, ratio, type,

and molecular properties of the collagen fibers and matrix within the mesoglea

are the main components in determining its functions within the coral colony.

1.3 Properties of collagen

Collagen is a key component of the mesoglea and of all metazoan extra-

cellular matrices (Exposito et al., 2010). It is one of the most abundant

proteins in animals, exhibiting a wide variety of forms and functions, providing

the major mechanical support for cell attachment and determining the shape

and form of tissues (Exposito et al., 2010). The collagen molecule has a

characteristic feature that is repeatedly noted in all metazoans (Garrone,

1998; Müller, 2003). It is composed of a long triple-helical domain in which

9

three collagen polypeptide chains are wound around one another to form a

rope-like superhelix, ~1.5 X 300 nm in size (Fratzl, 2003). The polypeptide

chains of the collagen molecule uniquely exhibit two features: glycine is every

third residue, generating a repeating (Gly-X-Y)n pattern; and a high

proportion of residues (ca 20%) are proline and hydroxyproline (Brodsky and

Ramshaw, 1997). This amino acid sequence allows the chains to form a right-

handed triple-helical structure, where all glycine residues are buried within the

core of the protein (Fig. 1). The residues X and Y are exposed on its surface

and function in the different collagen interactions (Nagarajan et al., 1999).

Figure 1. The collagen molecule, three collagen polypeptide chains (a) wound around one

another to form a rope-like superhelix, note Glycine residues (in brown) are buried within the

core of the protein (b). (From Molecular Cell Biology Freeman and Company ,2008)

11

Types of collagen differ from each other in the composition of the amino acids

in the polypeptide chains, and in the composition of chains within the

procollagen molecule (Lamande and Bateman, 1999; McLaughlin and Bulleid,

1998). The polypeptide chains can be either identical three α1 chains, as in

type III collagen or different, two α 1 chains and one α 2 chain, as in type I

collagen (Hulmes, 2002). The main fibrilar collagens are types I, II, III, V, and

XI (Kadler et al., 1996; Myllyharju and Kivirikko, 2001). Type I collagen is the

most abundant, being present in most vertebrate tissues, mainly in bone,

tendon and skin. Collagen types II and III are the second most abundant and

occur particularly in tissues exhibiting elastic properties, such as blood

vessels, internal organs, cartilage, inner ear, and central portion of the discs

between vertebrae (nucleus pulposus) (van der Rest and Garrone, 1991).

Other nonfibrilar collagens form networks (types IV, VIII, and X), occurring as

transmembrane proteins (types XIII and XVII), or form 11-nm periodic beaded

filaments (type VI) (Hulmes, 2002). Although all collagen types contain the

repeating sequence and fold into a characteristic triple-helical structure, they

are differentiated by the ability of their helical and non-helical regions to

associate into fibers, to form sheets, or to cross-link different collagen types

(Fratzl, 2003). These differences are responsible for the diverse functions of

collagen types in biological systems.

Collagen can present different formations, ranging from gelatinous to fibrous

rope-like structures. All of them are arranged hierarchically, wherein multiple

tropocollagen molecules form the collagen microfibrils/fibrilis, and multiple

collagen fibrils can form collagen fibers (Kadler et al., 1996) (Fig 2). The

collagen molecules in the fibril are arranged in a quasi-hexagonal lattice

11

lateral structure (Orgel et al., 2000), where collagen molecules are initially

assembled by polar, hydrophobic, and other non-covalent interactions, and

later by covalent cross-links (Ottani et al., 2002). Each collagen molecule is

offset by ~30 nm with respect to its lateral neighbors. This ~30 nm gap is

responsible for the fibrils possessing alternating differences in electron

density, with a 67 nm repeat that corresponds to the gap and the overlap

regions of the collagen molecules (Toroian et al., 2007). A microfibril is

thought to be the basic structural unit of the collagen fibril. Orgel et al. (2006)

demonstrated that each collagen molecule associates with its packing

neighbors to form a super-twisted, right-handed, pentameric microfibril that

interdigitates with its neighboring microfibrils to form a fibril. The final fibril can

be from 20 to 400 nm in diameter (Moeller et al., 1995). Collagen fibrils

provide the key to scaffolding structures from the nanoscopic to macroscopic

length scales, and are substantial constituents of skin, tendon, bone,

ligament, cornea, and cartilage as well as the cnidarian mesoglea (Fratzl,

2008; Vogel, 2003). The interaction of collagen fibrils with each other and with

the matrix surrounding them has a major role in shaping the morphology of all

metazoan extra-cellular matrices and connectives tissues (Fratzl, 2008).

12

Figure 2. Collagen structure, multiple tropocollagen molecules form the collagen

microfibrils/fibrilis, and multiple collagen fibrils form collagen fibers. (From Biology, Cambell

,1995)

The overall structural and the mechanical properties of collagenous materials

are largely derived from the water-retentive capacity of their fibrils and the

matrix surrounding them (Cameron et al., 2002). For example, at physiological

levels of hydration, the type I collagen fiber is comprised of about 30%

collagen and 70% water by volume (Toroian et al., 2007). This ratio can

change under progressive hydration, resulting in an increase in the fiber’s

diameter, but not in its length (Fullerton and Amurao, 2006). Connective

tissues, such as skin, cartilage, tendon, and blood vessels, are defined as

systems of insoluble fibrils and soluble polymers which have evolved to

absorb the stresses of movement and to maintain shape (Scott, 1975). The

insoluble fibrils are collagen, which is almost inextensible, and the soluble

polymers are mostly proteoglycans that swell in water (Vogel, 2003). This two-

element system enables the connective tissues to tolerate both pulling and

13

pushing (Fratzl, 2008). This is achieved by using the collagen fibrils to resist

pulling, while the proteoglycans press against the collagenous meshwork, in

order to resist compressive forces. For example, in tendons, which resist or

transmit tensile stresses, the fibrilar element predominates, whereas in

vertebrate cartilage or in the cnidarian mesoglea, which elastically absorbs

compressive forces and stores elastic energy (Scott, 1990; Thompson and

Kier, 2001), soluble polymers and water comprise the majority of the mass

(Vogel, 2003). Even small differences in the proportion of collagen fibrils and

matrix can significantly affect the tissue’s functional properties, and define two

classes of tendons: one that is strong and less flexible; and the other that is

flexible and functions as a spring (Ker, 2007). This association of fibrilar and

nonfibrilar collagens with other macromolecules and water, into organized

structures, can be observed throughout the entire diversity of metazoans

(Kadler et al., 1996).

1.4 Evolution and characteristics of collagens among invertebrates

Among the 21 types of collagen that have been described in humans, only the

fibrilar and the basement membrane type IV collagens are found in the

earliest branching multicellular animals (i.e.,Porifera and Cnidaria) (Exposito

et al., 2008).The existence of at least one fibrilar collagen and one nonfibrilar

collagen in these phyla indicates that the divergence between the collagen

families occurred early on in evolution. As the fibrilar collagen family has

relatively little evolved over time (Exposito and Garrone, 1990), the nonfibrilar

collagen found in the earliest branching multicellular animals might reflect two

evolutionary lines. One of these might have been the "exocollagens," such as

those attaching sponges to their substrate, the exoskeletons of cnidarians,

14

the cuticles of nematodes and the secreted collagens of mussels. The other

line might concern an internalization of such collagens, leading to the

differentiation of basement membrane and mesoglea (Exposito et al., 2002;

Exposito and Garrone, 1990). Reviewing the appearance of collagen in the

different invertebrate phyla discloses its evolutionary scenario and the

interrelationships among all metazoan groups.

The involvement of collagens in different vertebrate functions, such as the

construction of extracellular matrix and cell–matrix mediation, has already

been noted in sponges, where fine collagen fibrils are involved in the

construction of their mesohyl (Muller-Parker and DiElia, 1997). Notably, type

IV collagen, which is strongly associated with cell-adhesion, has been

described in both spongin (Exposito et al., 2008) and vertebrate basement

membrane (Aouacheria et al., 2006). This resemblance in collagen functions

between invertebrates and vertebrates is further noted in Cnidaria, where

ultrastructural and functional studies have revealed similarities between the

hydra- mesoglea and vertebrate basement membranes (Deutzmann et al.,

2000). Another example of the evolutionary role of collagen is related to the

association of collagen and calcium, as collagen takes a major part in the

construction of vertebrate skeleton in the form of calcium-phosphate-rich

collagen fibrils (Garrone, 1998). This was also found among Cnidaria, where

collagen plays a role in the formation of axial skeletons of sea pens (Ledger

and Franc, 1978) and sea fans (Kingsley et al., 1990).

15

While some invertebrate collagens present properties and functions that link

them to those found in vertebrates, others also present unique features not

known in the latter. For example, the shortest known collagens, named

minicollagens, are a family of unusually short molecules isolated from

cnidarians (Adamczyk et al., 2008), whereas the largest collagens are found

in the cuticle of annelid worms and are ca. eight times larger than most known

collagens (Gaill et al., 1991). Annelids that dwell near hydrothermal vents

were also shown to possess collagens with high thermal stabilities (Bris and

Gaill, 2007; Gaill et al., 1995). Collagens with unique biomechanical and

physical properties have also been found among some mollusks (Kier and

Smith, 2002; Thompson and Kier, 2001; Trueman and Hodgson, 1990). The

best studied collagen is the one produced by mussels, and which attaches to

the substratum by means of byssus threads. These threads are extracellular,

nonfibrilar and collagenous, secreted from the mussel’s foot (Bell and

Gosline, 1996). They function under tension as shock absorbers, being strong

and stiff at one end and pliable at the other (Waite et al., 2003). The distal

portion of these threads has a breaking strength comparable to that of a

tendon, while its proximal portion is approximately 20 times more extensible

(Bell and Gosline, 1996; Smeathers and Vincent, 1979). Invertebrate

collagens are not only unique in their ability to present a variety of properties,

but also in their ability to control and change these properties back and forth.

Mutable collagenous tissue occurs in a variety of echinoderms (Matranga,

2005), that can control the sliding rate of collagen fibrils when the tissue is

under tensile stress (Smith et al., 1981). This can rapidly and reversibly alter

the stiffness of their connective tissues, as opposed to connective tissues

16

such as tendons and ligaments that are generally regarded as passive and

inert materials (Szulgit, 2007). These examples indicate the large set of

different, and sometimes even exceptional functions, that collagen presents

among vertebrates and invertebrates (Heinemann et al., 2007). Although

considered a common structural protein in metazoans, the large degree of

polymorphism makes collagens one of the most studied protein families

(Exposito et al., 2002). Collagen occurrence in all invertebrate phyla has

made it a subject for study in a variety of both benthic and pelagic taxa

(Aouacheria et al., 2006; Helman et al., 2008; Tucker et al., 2011). The

diversity and complexity of invertebrate collagens may already exceed the

more extensively characterized vertebrate ones. Given that invertebrates

account for at least 95% of animal species, and that only some invertebrate

collagens have been characterized to date, one might expect a large

spectrum of unique collagens among them still to be found (Exposito et al.,

2002; Har-el and Tanzer, 1993).

This current study deals with the reef dwelling octocoral Sarcophyton

ehrenbergi (family Alcyoniidae). This zooxanthellate coral inhabits reefs

exposed to strong tidal currents and typhoons (Dai, 1993). Its congenerics

were found to be dioecious broadcasters, with onset of reproduction at the

age of 6-10 years in S. glaucum, or coinciding with 12-13 cm basal stalk-

circumference in S. elegans (Benayahu and Loya, 1986; Hellstrom et al.,

2010). S. ehrenbergi has a wide Indo–Pacific distribution, and is known for its

diverse content of natural products (Cheng et al., 2009; Fleury et al., 2000;

Look et al., 1986; Nguyen Xuan Cuong, 2008; Tanaka et al., 2005); these

17

include cembranoid diterpenes that, in therapeutically relevant assays, have

shown some cytotoxic, cancer chemo-preventative and anti

inflammatory potential (Konig and Wright, 1998).

1.5 Objective and aims

The overall objective of the present study was to identify and characterize the

nature of unique fibers found in colonies of Sarcophyton ehrenbergi. These

fibers are organized as bundles within the colonies and, when mechanically

isolated, may reach hundreds of microns in diameter and up to tens of

centimeters in length.

At the early stages of the study, two alternative hypotheses regarding the

nature of the fibers were addressed. The first argued that the fibers are

collagenous, while the second claimed that they are a semi-crystalline

polymer. The rationale behind these two hypotheses was derived from

previous studies on long fibers among invertebrates, such as the collagenous

byssus threads (Bell and Gosline, 1996) and the semicrystalline polymer

proteins of spider dragline silk (Simmons et al., 1996). Preliminary NMR and

amino acid analyses rejected the second hypothesis and accordingly dictated

the subsequent research. Additional hypotheses were tested regarding the

arrangement of the fibers and their function. It has been hypothesized that the

fibers are compactly arranged within the colony (i.e., folded or coiled) and not

linearly arranged in a stretched manner. Regarding the function of the fibers

in the colony, the working hypothesis posited that the fibers provide some

structural benefits to the colony as known for other collagen structures (Fratzl,

2008; Vogel, 2003).

18

Specifically, the current study dealt with the biochemical and structural

properties of the fibers of S. ehrenbergi, including their location and formation

at the tissue and cellular levels. In addition it evaluated the biomechanical and

physical properties of the fibers. The following questions were addressed:

What are the biochemical composition of the fibers and their spatial

arrangement?

Where the fibers are located and what is their three-dimensional structure

within the colony?

Where does the biosynthesis of the fibers take place?

What are the mechanical and physical properties of the fibers?

Answering these questions allowed me to examine the possible biological role

of the fibers in S. ehrenbergi colonies, and to gain new insights regarding the

evolution of collagen in metazoans. Studying the function of these fibers

within the colony can lead to a better understanding of how organisms

withstand environmental forces such as waves, currents, and tidal changes

(Vogel, 2003). In addition, the findings were compared to data from the

literature regarding known collagen fibers of invertebrates and vertebrates.

The fibers may also present additional morphological character that may

provide new insights into the relationships within and between the alcyoniid

genera Sarcophyton and Lobophytum. A molecular phylogenetic study of

these two genera identified a third distinct clade that includes a mix of nominal

species from each genus (McFadden et al., 2006). As this mixed clade

includes S. ehrenbergi, the study of its fibers has accumulated structural data

that may support the phylogenetic data. Elucidation of the biomechanical and

19

physical properties of the coral collagen fibers is also important in order to

assess the feasibility of utilizing them for biomedical applications.

21

2. Materials and Methods

2.1 Collection of colonies

Colonies of S. ehrenbergi were collected by SCUBA from several localities:

Dahalak Archipelago, Eritrea; Shimoni, Kenya; Kenting National Park, Taiwan

and Eilat, northern Gulf of Aqaba, Israel. Identification of the species was

facilitated by comparison to museum specimens deposited at the Zoological

Museum, Tel Aviv University (TAU). After collection, most of the colonies

were frozen to -200 C and shipped by air to TAU. Some colonies from Taiwan

were shipped alive to TAU.

2.2 Farming of colonies

Live colonies of S. ehrenbergi were maintained at TAU in a closed seawater-

system. The system comprised five PVC tanks, 1 m3 each, kept in a 30 m2

greenhouse that was protected from direct sunlight by a 50% shade net. Metal

halide lamps (Osram, HQI-BT 400W/D) provided extra lighting on occasions

of overclouding, when direct sunlight was absent for more than three

consecutive days. Each tank contained 800 L artificial seawater (Red Sea salt

©), 200 kg of live rocks obtained from Eilat and 1.7.-2.7 mm grain size coral-

sand (Pacific ©) on its bottom.

For algal grazing, each tank was provided with ~40 Trochos dentatus snails

and every 10 days their feces were removed from the bottom of the tanks,

together with ca 5% of the water volume, which was then replaced. Seawater

was circulated between the tanks by a 5,000 l per hour circulation pump

(JEBO), and was filtered through protein skimmers (JEBO 3500). Independent

21

water motion in each tank was generated by a centrifugal pump. An individual

heater (150 Watt) and a chiller (JEBO 2000) maintained the water

temperature in each tank (250 C, see ahead). The abiotic parameters in the

system were set as follows: salinity (35 ppt) and temperature (250 C) were

monitored daily, and the nutrient levels and pH were monitored weekly (nitrite

<0.05 ppm, nitrate <10 ppm, ammonia 0 ppm and 8.1-8.3 pH) (Sella and

Benayahu, 2010).

2.3 Extraction of fibers

Bundles of fibers were mechanically isolated from pieces (1-10 cm3), removed

from the polypary (the polyp-bearing part of the colony) of S. ehrenbergi

colonies (Fig. 4). The fibers were extracted from the tissue onto a revolved

polyethylene card (1x 8 cm) mounted on a low-speed electric motor (9-18

RPM). The card was removed from the motor and the fibers were cleansed of

cellular debris under a compound microscope using fine forceps, following 4-6

rinses in 70% ethanol. The fibers were kept rolled on the card in ethanol for

further experimental work.

2.4 Proton and carbon nuclear magnetic resonance analyses (NMR)

In order to characterize the main biochemical compounds composing the

fibers, NMR analysis was preformed at the School of Chemistry, Faculty of

Exact Sciences, TAU (August 2005 - August 2007). The NMR method is

based on the ability of a strong external magnetic field to create characteristic

resonance frequency when applied to structurally distinct sets of hydrogens in

a molecule. Fourier transform spectrometer operates by exciting all the proton

22

nuclei in a molecule simultaneously, followed by mathematical analysis of the

complex resonance frequencies emitted as they relax back to the equilibrium

state. The overlapping resonances signals that are generated as the excited

protons relax are collected by a computer and subjected to a Fourier

transform mathematical analysis. This analysis converts the complex time

domain signal emitted by the sample, into the frequency domain spectrum,

doing so it can acquire a complete spectrum within a few seconds (Arnold and

Marcotte, 2009).

For the purpose of the current study ca. 25 mg of fibers were removed from 6

colonies collected at different sites. The samples were hydrolyzed in 6 ml of

NHCL overnight at 110 °C. The acid was then removed under vacuum and

the residue dissolved in D2O (0.5 ml). Samples were measured in 500 MHz

and 100 MHz NMR machines (Bruker ARX500, ARX400).

2.5 Amino acid analysis

In order to characterize the protein that composes the fibers, amino acid

analysis was preformed at the Department of Chemical Research Support,

Weizmann Institute of Science (January 2006). This method enables

determination of protein quantities and provides detailed information regarding

the relative amino acid composition and free amino acids (Bütikofer et al.,

1991). The procedure includes hydrolysis, following by a separation, detection

and quantification, which give a characteristic profile for a protein, often

sufficient for its identification.

23

Peptide hydrolyzates were achieved using ophthalaldehyde, 3-

mercaptopropionic acid (OPA/MPA), and 9- fluorenyl-methyl chloroformate

(FMOC). Sensitive detection and separation of hydrolysed samples (amino

acids) were done by HPLC. The pre-column preparation was fully automated

and had a detection range of 100-3000 pmoles, thus requiring only a small

sample (1 µl). Three samples of isolated collagen fibers (ca. 5 µg each) from

samples collected at different collection sites were analyzed using Waters

PicoTag Work Station for gas phase Hydrolysis and Hewlet Packard 1090

HPLC equipped with a diode array detector and an auto injector with a PC

based Chemstation database, utilizing Amino Quant chemistry.

2.6 Light and electron microscopy

In order to examine the microstructural features of the collagen fibers, light,

scanning, and transmission electron microscopy (SEM, TEM) were used. For

visualization of fibers in the tissue and of isolated fibers, samples were

removed from colonies that had been earlier preserved in 4% glutaraldehyde

in seawater. The samples were decalcified in a mixture of equal volume of

formic acid (50%) and sodium citrate (15%) for 20 minutes (twice), and then

placed back in 4% glutaraldehyde. Samples for light microscopy were rinsed

with distilled water, and embedded in 2 % agarose (50 °C), or in high melting

point paraffin. This procedure was conducted in order to maintain the natural

orientation of the collagen in the colony and in the isolated fiber bundles while

sectioning them. Following solidification, rectangular pieces, closely fitting

around each sample, were cut out and transferred to 70% ethanol. After

dehydration through a graded series of ethyl alcohol, the samples were

24

embedded in paraffin. Cross and longitudinal sections, 5-8 μm thick, were

prepared using MIR microtome (Thermo Fisher Scientific, Waltham, MA).

Sections were routinely stained in Hematoxylin – Eosin and additionally in

Masson blue which stains collagen, Van Gieson- elastin and Alcian blue-

mucopolysaccharides and glycosaminoglycans (Ross and Wojciech, 2006)

(Ross and Wojciech, 2006).

Samples for SEM and TEM were decalcified (see above) and later dehydrated

through a graded series of ethyl alcohols. Samples for SEM were fractured

using a scalpel blade in order to expose the polyp cavities, before being

critically point-dried with liquid CO2, gold coated, and examined under JEOL

JSM 840A SEM operated at 25 kV. Material for TEM was embedded in Epon

and the sections were stained with both uranyl acetate and lead citrate.

Glycoproteins were detected by using sodium tungstate and cupromeronic

blue staining (Scott, 1990). Negative staining was employed for studying fibrils

that were detached from fibers by sonication at 30 KHz for five minutes (PCI

1.5) (Ortolani and Marchini, 1995). TEM was carried out with Jeol 1200 EX

electron microscope.

2.7 Graphic analysis

Image-J software (National Institutes of Health, USA) was used for analyzing

gray values distribution and calculating areas on selected areas in

micrographs and histological images. The software was used to create gray

value intensity histograms that calculated the color intensity distribution along

25

fibrils in TEM micrographs. This was done in order to locate repeated staining

patterns along the fibrils. The color intensity histogram displayed a two-

dimensional graph of the intensities of pixels along rectangular selections (or

line selections wider than one pixel) within the image. The obtained graph

featured a ‘column average plot’, where the X-axis represented the horizontal

distance through the selection, and the Y-axis the vertically averaged pixel

intensity. In order to define a tested area on a TEM micrograph of a fibril, the

image was set as 8-bit gray scale image and a rectangular border, 200-500

nm long, was placed over the fibril (14 samples from 9 different isolated

fibrils) (Ferreira and Rasband, 2011).

2.8 Wide-angle X-ray diffraction

In order to characterize the molecular structure and peptide arrangement of

the collagen, wide-angle X-ray diffraction study was performed on the fibers at

the Department of Molecular Microbiology and Biotechnology, TAU

(November, 2008). To obtain X-ray diffraction data, samples of isolated fibers

were dried at room temperature for 15 minutes and stretched along a

rectangular slit in a brass sample holder, secured on either side by

cyanoacrylate adhesive (3M). The experiments were performed on Rigaku R-

axis IV++ image-plate detector mounted on a Rigaku RU-H3R rotating anode

generator with Cu Kα radiation focused by Osmic confocal mirrors. The

detector sample distance was 100 mm, and a 1.5418 Angstroms wavelength

was used, following Pazy et al. (2002).

26

2.9 Magnetic Resonance Imaging (MRI)

In order to study the distribution of the fibers within the colony, an MRI study

was conducted at the Department of Neurobiology, TAU (September –

November, 2010). Four small colonies of S. ehrenbergi with a polypary-

diameter of 3-5 cm were scanned by 7T/30 MRI scanner (Bruker, Germany),

equipped with a gradient system of 400 mT/m. Prior to the test the colonies

were anesthetized by 4-hr titration of saturated MgCl2 solution (Häussermann,

2004). Throughout the scan, the colonies were kept in 50 ml PVC test tube

filled with saturated MgCl2 seawater at 23° C. For excitation, a body-coil

(outer/inner diameter of 112/72 mm) was used and a quadrate coil (10 mm

diameter) served as a receiver. The MRI protocol included Gd-enhanced fat-

suppressed T1-weighted imaging and DTI. The total MRI protocol lasted

120 min, in order to obtain an image with maximal resolution.

2.10 Biomechanical studies

In order to evaluate the biomechanical properties, bundles of fibers were

isolated from colonies and stored in 70% ethanol and shipped by air to Friday

Harbor laboratories, Washington, USA (June, 2008). Prior to the experiment,

samples were placed for one hour in fresh water (FW) at room temperature in

order to rehydrate the samples to their original state. Single fibers were

isolated from the bundles using fine forceps under a dissecting microscope.

The fibers were photographed (X 1000) with a camera attached to a light

microscope (Nikon, DN 100), and their length and diameter was digitally

measured by ImageJ software (Fig. 3a). A fiber was attached by cynoacrylate

glue at one end to a stainless steel tensometer beam with a half bridge

27

formed by two semi-conductor strain gages (1gr=7.45V, max 10 gr). The other

end the fiber was fixed to a stainless steel beam maneuvered by micrometer

(Fig 1b) see Kasapi and Gosline (1999). During the installation, measures

were taken to minimize axial stretching of the sample and to keep the sample

moist. The sample was installed in an experimental chamber containing

distilled water, and was immersed in relaxed (un-stretched) state for ca. 30

minutes before testing to insure rehydration (Gentleman et al., 2003; Kasapi

and Gosline, 1999).

Figure 3. The soft coral Sarcophyton ehrenbergi. Biomechanical experiment set-up. a. Fibers

under light microscope (100x, oil immersion). b. Tensometer on stage of dissecting

microscope, note stainless steel beam maneuvered by micrometer on one side of the

experimental chamber and stainless steel tensometer beam on the other.

Prior to the experiments, the force transducer beam deflection was measured

by pulling a thin (0.5 mm) non-flexible copper wire in known increments. The

calculated beam deflection was subtracted from all fiber displacement results

in order to obtain the real displacement. In the experiments, undulated fiber

a b

28

was extended until becoming straight but not stretched, and its reference

length was measured by caliper in mm (L initial). In order to locate L0, the

point where internal material alignment makes the material respond in a

similar way to deformation (Vogel, 2003), fibers were repeatedly stretched to

5% of their initial length (i.e., preconditioning cycles). Preconditioning to 5% (3

cycles) was performed in each experiment before the samples underwent

elongation profiles of load-unload (4 cycles) or load to failure, and L0 was

measured. Volt output was gained x1000 and reading was carried out with a

voltmeter (0.001 precision). Micrometer maneuvering and data recording were

performed by hand in 100 µM increments, and force (N) was calculated.

The results of the experiment included the fiber’s extension (µm) and load

bearing (N).

To analyze the results in a manner independent of the sample dimensions,

the elongation and load were respectively transformed to strain (e) and stress

(s). s=F/A e= ΔL/L0

Where F [in Newton] is the load, and A= πR² the cross-sectional area of the

fiber following Vogel (2003).

Hysteresis was observed in the stress-strain curve, as the area of the loop

being equal to the energy lost during the loading cycle.

Viscoelasticity was observed as the property of the material to exhibit both

viscous and elastic characteristics when undergoing deformation.

Modulus of elasticity was defined as the slope of the stress–strain curve in the

elastic deformation region: λ= s/e where lambda (λ) is the elastic modulus; s

is stress and e is strain. λ units are Pascal.

29

Strength was defined as the maximal stress achieved when a sample is

loaded until it breaks.

Extensibility was defined as the maximal strain achieved when a sample is

loaded until it breaks.

Data analysis was performed using Microsoft Excel.

In order to characterize the mechanical properties of a fibril and its attachment

force to the fiber, in-situ SPM-TEM testing was performed at the Center for

Nanoscience and Nanotechnology, TAU (February, 2010). The TEM-SPM

(Nanofactory Instruments Inc., Dallas, U.S.A) consists of a piezo tube for fine

motion, and a geared stepping motor for rough z-motion. The TEM-SPM was

inserted into a Philips CM200 field emission gun TEM, and AFM cantilever

took force measurements by directly measuring displacement of the AFM tip.

A new experimental procedure was developed in order to utilize the TEM-

SPM system for the biomechanical fibril-fiber interaction study. For the

sample preparation, fibers were attached in parallel to a TEM grid by

cynoacrylate glue on both sides. After he adhesive had set, the TEM grid was

cut in the middle using electric pliers, creating two semi-circular unites with a

number of protruding free-cut fiber ends. Each experiment used one semi-

circular TEM grid that was inserted into the TEM. TEM operational adhesive

(Nanofactory Instruments Inc., Dallas, U.S.A) was applied to the free end of

the AFM cantilever, in order to hold protruding fibrils from the cut ends. Four

samples were tested during this study. Force measurements and continuous

digital photography were recorded during the experiments (Regan et al.,

2004).

31

2.11 Thermogravimetric analysis and differential scanning calorimetry

In order to measure the weight changes in the collagen and to determine its

thermal stability, Thermogravimetric Analyzer (TGA) and Differential Scanning

Calorimeter (DSC) were carried out at the Wolfson Applied Materials

Research Centre, TAU (March, 2010). For this purpose TA instruments

module 910 and System Controller 2100 were used. For TGA measurements,

fibers in 70% ethanol were transferred to three drying treatments: room

temperature for 15 minutes and 24 hours, as well as 24 hours in vacuum

(250C). For the TGA measurement the samples were sealed in a glass vessel

and placed in a sample compartment that was continuosly flushed with dried,

pre-purified argon. The scan rate of TGA runs was 0.50C/min, following

Golodnitsky et al. (2003).

DSC was used for measurements of heat-flow and temperatures associated

with transition of the soft coral collagen from organized structure to

unorganized state. For the DSC measurements, vacuum-dried samples of 5-

12 mg (24 hours), were hermetically encapsulated in aluminum pans. The

fibers were subjected to a sinusoidal temperature ramp, superimposed on a

linear temperature ramp in order to provide data on reversing and non-

reversing characteristics of the thermal events. DSC runs were recorded at a

scan rate of 5 -100 C per minute up to 300° C (Leikina et al., 2002).

31

3. Results

3.1 Biochemical and structural properties of isolated fibers

Tearing apart the polypary of S. ehrenbergi colony revealed numerous

bundles of fibers that could be pulled from the tissue to a length of at least

one order of magnitude higher than the colony’s diameter (Fig. 4).

Figure 4. The soft coral Sarcophyton ehrenbergi. Mechanically isolated fibers from the

polypary. Note the large number of bundles of fibers and their length.

3.1.1 Proton and carbon nuclear magnetic resonance (NMR)

Proton and carbon NMR analysis revealed spectra that are characteristic for

hydrolyzate of a peptide that is a mixture of amino acids, thus indicating the

proteinous nature of the fibers (Fig. 5).

32

3.1.2 Amino acid analyses

Amino acid analysis further confirmed the presence of amino acids and

revealed high concentration of glycine, proline and hydroxyproline in a ratio

corresponding to a collagenous protein (Fig. 6) (Fratzl, 2008). Table 1

presents the release time of amino acids from the mobile phase gradient in

the chromatographic column (release time in minutes: RT), the intensity of the

observed peak in fluorescence units, its area, and the calculated amount in

picomol. Eight out of the 27 peaks noted in the sample were not recognized

as known amino acids (see Discussion).

33

Figure 5. The soft coral Sarcophyton ehrenbergi. NMR spectroscopic profile of fibers

featuring the characteristic spectrum of hydrolyzate of peptide comprised of an amino acid

mixture.

34

Figure 6. The soft coral Sarcophyton ehrenbergi. Amino acid analysis of fibers featuring

collagen-related amino acids. Glycine, Proline, and Hydroxyproline are indicated.

35

Table 1. The soft coral Sarcophyton ehrenbergi. Characterization of amino acid peaks. Note

eight unrecognized peaks (9, 12, 14, 16, 17, 21, 22 and 27).

No. Name RT Area Height Amount (pmol) % from known AA

1 Hpro 11.797 1449440 98789 83.674 4.15

2 AMQ 12.608 210420 10577 43.312 2.15

3 Asp 13.625 2201385 143512 168.082 8.34

4 Ser 15.347 1465071 97687 87.668 4.35

5 Glu 16.013 2789428 177478 191.959 9.52

6 Gly 17.494 7795628 423228 501.615 24.89

7 His 18.262 465666 26694 18.943 0.94

8 NH3 20.05 4225440 221045 149.948 7.44

9 21.306 80331 7356

10 Arg 21.666 2435609 222943 107.777 5.35

11 Thr 21.975 1571415 126517 69.217 3.43

12 22.815 56598 7125

13 Ala 23.115 3024957 238777 130.905 6.49

14 24.387 28980 1865

15 Pro 25.043 1105715 101985 102.471 5.08

16 26.748 22910 2261

17 27.852 60417 7533

18 Tyr 28.356 743866 91521 30.726 1.52

19 Val 29.306 3534428 356606 79.999 3.97

20 Met 29.788 1366922 147856 39.482 1.96

21 30.378 52533 5555

22 31.133 25901 1890

23 Lys 31.867 1399282 158919 59.693 2.96

24 lle 32.46 2794982 294791 49.841 2.47

25 Leu 32.884 3700211 392968 66.24 3.29

26 Phe 33.72 2419376 260384 33.978 1.69

27 34.587 85143 7256

Sum 2015.53 100.00

3.1.3 Light and electron microscopy

Histological staining of isolated bundles of fibers stained with Masson Blue,

Van Gieson, and Alcian blue revealed a packed arrangement of fibers

wrapped around each other. Two to three fibers coil together to form a rope-

like structure, and a number of such structures form a bundle (Fig. 7a, b). This

feature was also noted when isolating a single fiber using forceps for the

biomechanical studies (see below). Isolated relaxed collagen fibers are a-

cellular, almost circular in cross-section, with a diameter of 9 ± 0.37 µm

(n=166 fibers), and feature a wavy course (Fig. 7). Both Masson Blue and Van

Gieson stains stained the fibers homogenously, and confirmed their

36

collagenous nature (Fig. 7a, b, c). The blue (Masson Blue) and the red (Van

Gieson) stains revealed the same degree of coloration on all longitudinally-

sectioned fibers, as well as on the cross-sectioned ones. However, the degree

of coloration differed between the two types of sections. Cross-sectioned

fibers showed darker coloration than longitudinally-sectioned ones for both

Masson Blue and Van Gieson stains. Elastin, which is normally stained black

by the Van Gieson (Ross and Wojciech, 2006), was not found in either the

cross-or longitudinal-sections (Fig. 7c). Alcian Blue staining of isolated fibers

(Fig. 7d) did not generate homogeneous staining. The fibers did not reveal the

typical blue color associated with this staining (Ross and Wojciech, 2006),

and therefore there was no conclusive evidence regarding the presence of

mucopolysaccharides or glycosaminoglycans in the fibers. Only the agarose

that was used for mounting the collagen fibers was stained blue, and could be

seen between the fibers (Fig. 7d).

37

Figure 7. The soft coral Sarcophyton ehrenbergi. Histological cross-and longitudinal-sections

of stained isolated fiber bundles. a. Cross and longitudinal sections, Masson Blue confirms

the collagenous nature of the fiber by staining them blue, note the fibers wavy course ; b.

Longitudinal sections, Masson Blue, note a number of fibers (in blue) coiled together; c.

Cross-and longitudinal-sections, Van Gieson confirms the collagenous nature of the fiber by

staining them red-brown; d. Cross-and longitudinal-sections, Alcian Blue stained blue only the

agarose between the fibers.

38

TEM micrographs of cross and longitudinally sectioned fibers revealed a

parallel arrangement of thinner fibrils organized in a packed structure (Fig. 8).

Fibrils in longitudinal sections showed a repeated pattern of dark and light

banding perpendicular to the fibril axis and were not homogenously stained.

Cross-sectioned fibers featured a packed arrangement of fibrils, although

there was an unstained area between adjacent ones. The width of fibrils in the

longitudinal-sections was 15.9± 3.11 nm (n=15 fibrils in 3 fibers) and their

diameter in the cross-sections was 15.16± 3.41 nm (n=17 fibrils in 6 fibers),

according to microscopic image measurements. Fragments of fibrils isolated

by sonication and negatively stained featured a diameter of 18.9 ± 2.45 nm

(n=41 measurements on 27 isolated fibrils, Fig. 9a) as well as a repeated,

dark and light banding perpendicular to the fibril’s axis. Examination of this

banding along the fibrils through creating a color intensity distribution,

showed dark and repeated bands every 65-70 nm, with some lower

amplitude (less dark) repeatable bands inside the 65-70 nm zone (Fig. 9b).

The negative staining of isolated fibrils also revealed a parallel arrangement of

thinner sub-units ~2.5 nm wide, organized in a packed structure (Fig. 9c).

This pattern was also noted by TEM micrographs of the collagen fibril

protruding from the end of a fiber, which featured an uneven surface area

(Fig. 9d).

Cupromeronic Blue staining (CB) colored the fibers in a strong dark blue

visible to the naked eye. Longitudinal-sections in CB-stained samples

revealed a parallel arrangement structure of packed fibrils surrounded by a

dense proteoglycan matrix. This matrix seemed to be distributed evenly

39

between the fibrils and along the fiber (Fig. 10). When comparing the TEM

micrographs of longitudinal-sectioned fibers both with and without CB (Figs. 6,

8), it is evident that the CB labeled the areas between adjacent fibrils within

the fibers.

Figure 8. The soft coral Sarcophyton ehrenbergi. TEM micrographs of sectioned fibers. a.

Cross-section, b. Longitudinal section. Note parallel fibrilar structure.

41

Figure 9. The soft coral Sarcophyton ehrenbergi. Micrographs of fibrils and graphical

analysis. a. TEM micrograph of negatively stained isolated fibril, note bright horizontal bands

along fibril. b. Intensity histogram of a fibril section (yellow), major bands are ca 70 nm (dark

and red arrows), lower amplitude banding is also noticed inside the 70 nm unit (area under

red arrows). c. Negatively stained isolated fibril with organized pattern of elongated subunits

~2.5 nm wide. d. TEM micrograph of collagen fibril protruding from sectioned fiber with an

uneven surface.

41

Figure 10. The soft coral Sarcophyton ehrenbergi. TEM micrograph of longitudinal section of

isolated fiber with parallel arrangement of packed fibrilar structure and dense proteoglycan

matrix between fibrils (in black, marked by arrow).

42

3.1.4 Wide-angle X-ray diffraction

Wide-angle X-ray diffraction patterns recorded in the fibers revealed a strong

intermolecular function perpendicular to the helix layer line, just beyond the

salt ring. This was an indication of a lower resolution series of meridional

reflections of the molecular packing, which confirmed the fibrilar nature of the

collagen. The fibrilar packing function comprised fibrils aligned 16-17 nm

apart, with a periodicity of 66 nm. The helix pitch was approximately 2.86

angstroms and consisted of high proline content, with a molecular packing

function of ca 12 angstroms (Fig. 11). A significant water background was

noted, covering the first order.

Figure 11. The soft coral Sarcophyton ehrenbergi. Wide-angle X-ray diffraction of dry fibers,

helix layer lines and meridional reflections. Note significant water background (arrow) and the

different rings which are meridional reflections of the molecular packing. Darker field in central

part is due to rectangular slit in the sample holder which clasped the fiber.

43

3.2 Location, distribution and formation of fibers within the colony

3.2.1 Light and electron microscopy

Histological cross- and longitudinal sections from the polypary of S.

ehrenbergi revealed an accumulation of organized collagen fibers along the

gastrovascular cavity of the polyps in six out of the eight mesenteries. The

gastrodermal cells of these mesenteries create a ridge with a tube-like

structure along the free end of the mesenteries which encompass the coiled

fibers (Fig. 12a-c). This feature is noted in cross-sections in the basal (lower)

part of the pharynx and can be traced by serial cross sections down to the

base of the gastrovascular cavity (Fig. 12a-c). SEM micrographs of cross- and

longitudinal sectioned polyp cavities after mechanical tearing, showed fibers

of approximately nine µm in diameter, extending from the gastrodermal tube-

like structure along the free end of the mesenteries (see Fig. 12d-f). The fiber

had an uneven surface area, and it still featured a helical wavy structure, even

when it was removed from the heavily coiled packing within the mesentery.

TEM micrographs of gastrodermal cells of the mesenteries surrounding the

fibers revealed a large number of striated vesicles (Fig.11a-c). They were

found mostly within the gastrodermis of the mesentery adjacent to fiber

bundles (Fig.11d). High numbers of vesicles were also noted in cell clusters

found in the mesoglea (Fig. 14a, b). These cell-containing vesicles were

located near the sites of the decalcified sclerites, which were surrounded by a

fibrous structure. A fibrous sheath surrounded the remained debris (Fig. 14f)

and was comprised of fiber-layers. Their structure differed from the mesoglea,

which was comprised of unorganized fibrils embedded in a large volume of

44

matrix (Fig. 14g-i). As opposed to the fiber bundles found along the

mesentery, the fibrous sheaths seemed to be made of a three-dimensional

network of layered fibers, and were less dense than the parallel arrangement

of fibrils of the mesenterial collagen fibers.

Histological cross-sections from the stalk of S. ehrenbergi revealed an

accumulation of organized collagen fibers in gastrodermal cavities/canals that

extend into the stalk (Fig. 15). The gastrodermal cells of these gastrodermal

cavities/ canals, create a mesentery-like structure which encompasses a

fibrous collagen arrangement. These cavities/ canals were found to be smaller

and more densely arranged in the periphery of the stalk than at its center.

45

Figure 12. The soft coral Sarcophyton ehrenbergi. Location, arrangement, and structure of

collagen fibers within a polyp. a. Polyp morphology with a cross-section in the gastrovascular

cavity; note that six out of eight mesenteries contain collagen fibers (marked in black) . b.

Histological cross-section of gastrovascular cavity with fiber bundles (in blue) within six out of

eight mesenteries. c. Histological cross-section of collagen fibers within mesentery, note

packed rope-like arrangement of bundles of fibers and gastrodermal cells surrounding them.

d. SEM micrograph of fibers emerging from mesentery within polyp cavity; note helical

structure and fibrilar texture of fibers. e. Fiber bearing mesentery; note tube-like structure

formed by gastrodermal cells and extended fiber. f. Collagen fiber extended from the

mesentery; note the heavily coiled fiber (right side) as it loosens (left side).

46

Figure 13. The soft coral Sarcophyton ehrenbergi. TEM micrographs of gastrodermal cells

within mesentery. a Cross-section of fibers within a mesentery; note gastrodermal cell (GC)

and fiber bundle (FB). b Cross section of gastrodermal cell with fibrous vesicles (FV). c

Fibrous vesicles (FV). d Fibrous vesicles adjacent to inner side of cell membrane (marked by

arrows on both sides) bordering collagen bundle; note collagen fibrils.

47

Figure 14. The soft coral Sarcophyton ehrenbergi. Microscopy of Mesoglea. a Longitudinal-

sections of polypary; note polyps cavities (PC), clusters of cells within mesoglea (CM), and

space left after decalcification of sclerites (SS). b Cross-section of mesoglea (Masson Blue);

note strong coloration of fibers on circumference of decalcified sclerite (arrow). c TEM

micrograph of cells clusters within mesoglea (M) with fibrous vesicles (FV). d, e Fibrous

vesicle. f Fibrous sheath (CF) surrounding decalcified sclerite. g, h Layered fibrous sheath. i

Unorganized arrangement of collagen fibrils within the gelatinous mesoglea.

48

Figure 15. The soft coral Sarcophyton ehrenbergi. Location, arrangement, and structure of

collagen fibers. Histological cross-section of the colony stalk. a. Gastrovascular cavity/ canals

(GC) arrangement within the stalk; note the ectoderm (ECT) and the increase in size of GC

towards the center of the stalk. b. Gastrovascular cavity/ canals with 2-3 mesentery-like

structures which encompass a fibrous collagen arrangement (CL); note CL in both intact and

damaged mesenteries-like structures (arrows). c. Gastrovascular cavity/ canals at the center

of the stalk. d. Gastrovascular cavity/ canals at the peripheral layers of the stalk.

49

3.2.2 Magnetic resonance imaging (MRI)

Serial MRI cross-sections of Sarcophyton ehrenbergi colonies, starting from

the surface of the polypary down to the stalk, revealed the fiber arrangement

within the colony. The fibers extent from the polypary to the colony base, and

were traced as originating from all the polyps on the polypary, both top and

periphery, and progressing through the stalk to the colony base (Fig. 16).

Cross-sections of the stalk showed that the fibers are not evenly distributed

within the stalk, but run along the stalk peripheral layers.

Figure 16. The soft coral Sarcophyton ehrenbergi. Magnetic resonance images of colony. a.

Sarcophyton sp. colony (3 cm in diameter), the numbered horizontal lines correspond to

levels of imaging within colony. b. Images of 50 µm cross-sections of colony (1-16), ranging

from the polypary (1) to the basal part of the colony(18). c. Magnification of three images (1, 6

and 16), collagen bundles appear black and marked by arrows; note fibers running from

polyps with different spatial orientation (1 vertical and 6 horizontal) to periphery of stalk (16).

51

3.3 Biomechanical and physical properties of the fibers

3.3.1 Biomechanical properties of isolated fibers

Preconditioning: L0 was obtained from strain-stress curves of preconditioning

cycles (Fig. 17). Average difference between L intial and L0 was 0.03 ±

0.0094 (n=7 fibers).

Load-unload cycles: From strain-stress curves obtained from fibers that were

repeatedly stretched to 15% of their initial length (3 cycles of loading-

unloading), the viscoelastic character of the samples was evident (Fig. 17).

This was defined by a distinct hysteresis loop and decreased stress values

for the same strain between consecutive cycles. Average hysteresis for the

first cycle was 41.2595±15.5% (n=14 fibers). It was evident that the overall

response of the fibers was not a linear one. However, it seems that starting

from a certain strain level, there was a linear relationship between stress and

strain. Linear regression at the high range of strain (7% - 15%), showed a

very good correlation (R2=0.999). No correlation was found between slope to

sample length (p>0.05). The estimated slope was 0.5 ±0.1 GPa and

represents the stiffness of the sample.

Load to failure: From the load to failure strain-stress curves of 12 tested

fibers, it was evident that the overall response was not linear (Fig. 17). Linear

regression at the high range of strain (8 - 19.4%) revealed highly significant

correlation (R2=0.999), but no correlation was obtained between the slope and

fiber length (p>0.05). The estimated slope is 0.44 ± 0.1 GPa and indicates the

stiffness of the fiber. Average stress to failure was 55.6 ± 11.7 MPa and

average elongation under stress was 19.4±4.27%.

51

Figure 17. The soft coral Sarcophyton ehrenbergi. Mechanical properties of isolated collagen

fibers. a. Representative stress-strain curve of preconditioning cycle. b. Representative

stress-strain curve of loading-unloading cycle (E= 0.9 GPa). c. Representative stress-strain

curve of load to failure.

52

3.3.2 Mechanical characterization of fibrils with in situ SPM-TEM

A force-distance curve obtained under loading-unloading cycle of four fibrilis

(protruding from 4 fibers), showed elastic characters such as the hysteresis

loop and the decreased force values for the same distance between 0-250 nm

(loading) and 250-0 nm (unloading) (Fig. 18). Linear regression of both

loading and unloading curves of one cycle (60-200 nm) revealed a highly

significant correlation (R2=0.9845). An estimated slope of 0.036 ± 0.015 µN

(n=4) was noted in both curves, representing the stiffness of the fibrils.

Repeated loading-unloading cycles of the same fibril were not performed

because of the difficulty in stabilizing the fiber stable on the TEM grid as the

fibril was being pulled, and in eliminating fibril sliding within the fiber over time.

Furthermore, it was not possible to use the AFM cantilever tip for forces

higher than 8-9 µN because of a failure of the tip adhesive to hold the fibril

under such forces (Fig. 19). Therefore, a load to failure curve of a single fibril,

or the needed force for detachment from the fiber, could not be obtained.

53

Fibril- Fiber Desplacment

-2

-1

0

1

2

3

4

5

0 20 40 60 80 100 120 140 160 180 200

Distance (nm)

Fo

rce

(µ

N)

Figure 18. The soft coral Sarcophyton ehrenbergi. Mechanical properties of non- isolated, in

vivo collagen fibril. A representative force-distance curve of loading- unloading cycle.

54

Figure 19. The soft coral Sarcophyton ehrenbergi. Mechanical characterization of non-

isolated fibril with in-situ SPM-TEM. a. AFM cantilever tip (black) approaches a cross-section

of a collagen fiber with a protruding fibril (fibril marked by arrow). b. AFM cantilever tip

connected to fibril during a load-unload cycle. c. Magnification of tip-fibril connection area;

note folded end of fibril on adhesive tip (arrow). d. Adhesive failure as force over nine µN is

applied; note adhesive residue on the fibril end, and damage to the tip.

55

3.3.3 Thermogravimetric Analysis (TGA)

TGA of fibers stored in 70% ethanol and then dried at room temperature for

15 minutes, showed ~30% loss of weight as they were heated to ~1000C. This

weight loss was also noted in fibers maintained at room temperature for 24

hours (~13%). Even those maintained in a vacuum for 24 hours showed ~7%

weight loss along the heating profile (Fig. 20). These results led us to use

vacuum dried fibers in the following DSC analysis, in order to minimize the

masking of the enthalpy point.

3.3.4 Differential Scanning Calorimetry (DSC)

The DSC technique showed that the collagen fibers present two suspected

enthalpy areas, one at a range of 42-540 C and one at a range of 61-740 C

(Fig. 21a). The 42-540 C area peaked at 48.70 C and showed much lower heat

consumption than the 61-740 C area, and a relatively gradual change in heat

flow that did not yield a distinctive peak (Fig. 21b). In contrast, the 61-740 C

area showed large heat consumption that peaked at the distinctive

temperature of ~67.80C, and is considered the enthalpy point/ melting

temperature of the octocoral collagen.

56

Figure 20. The soft coral Sarcophyton ehrenbergi. Thermogravimetric analysis (TGA) of

isolated fibers. Weight changes along heating profile of three different fiber preparations:

fibers dried at room temperature for 15 minutes (red); for 24H (blue) and in vacuum 24H,

250C (green). Note that even vacuum-dried fibers showed 7.383% weight loss when heated

to ~1000C.

7.383% (0.1689mg)

tubes dried in vacuum.

115.96°C

tubes dried 24h at RT on filter pap in air atmos.

12.95% (0.2464mg)

tubes as is

30.40% (1.723mg)

40

60

80

100

120

Wei

ght (

%)

0 50 100 150 200 250 300 350

Temperature (°C)

––––––– collagen vac dried tubes.001– – – – Collagen-1 24h R-airTGA.001––––– · Collagen-1-TGA.001

Universal V2.6D TA Instruments

57

Figure 21. The soft coral Sarcophyton ehrenbergi. Differential scanning calorimetry (DSC) of

isolated collagen fibers. a Two heat consumption areas along heating profile: at 42-540C and

at 61-740C (marked). Denaturation occurred at ~ 67.8°C (arrow). b Detailed DSC of 42-54

0C

heat consumption areas that is rejected as the denaturation area; note the gradual change in

heat flow, and the lack of distinctive peak.

58

4. Discussion

Prior to the current study, the fibers of the soft coral S. ehrenbergi have not

been noted in the literature. These fibers were noticed while collecting

colonies from the reef, when their polypary was torn and the fibers were

consequently exposed. The fibers could then be pulled out from the tissues to

arm’s length while still remaining intact. These observations intrigued me,

leading me to seek answers to the questions that comprise the core of this

study: what are the fibers made of, and what is their possible function?

Elucidation of the molecular structure of these fibers and their physical

properties will conduce to assessing the feasibility of using them for myriad of

uses, from biomedical applications, to coral taxonomy.

4.1 Proton and carbon NMR and amino acid analyses

NMR analysis classified the fibers as a protein (Fig. 5). Amino acid analysis

revealed their collagenous nature, mostly due to the high concentration of

glycine, proline and hydroxyproline (Fig. 6). The amino acid composition

demonstrated 27 distinct peaks, including a high concentration of aspartic

acid, glutamic acid, alanine and arginine. Overall 19 peaks were recognized

and quantified, while eight could not be identified (Table 1). Twenty-two amino

acids are naturally incorporated within polypeptides and termed proteinogenic

or standard amino acids (Fratzl, 2008). These amino acids were identified by

the amino acid analysis (see above). It is assumed that the eight unidentified

peaks represent additional amino acids, which are among those found in

marine organisms and known as non-proteinogenic amino acids (NPAA)

(Nelson and Cox, 2000). The latter are formed by post-translational

59

modification, or as intermediates in the metabolic pathways of standard amino

acids (Curis et al., 2005). For example, hydroxyproline is a common NPAA

that plays a key role in collagen stability by permitting the sharp twisting of its

helical structure (Brinckmann et al., 2010). Although the complete sequence

of amino acids of the soft coral collagen is still unknown, it is suggested that

the unidentified peaks may contribute to its properties. Changes in the amino

acid composition of a protein can improve its metabolic and biomechanical

performance (Fratzl, 2008; Jackson et al., 2006). In collagen fibers, different

intermolecular cross-links between amino acids prevent slippage under load

and determine its mechanical properties (Fratzl, 2008). Animal collagens,

ranging from sponges to humans, are predominantly cross-linked by lysyl

oxidase, although some alternative mechanisms can be found in marine

organisms. For example, byssus threads of mussel bivalves feature a variety

of mechanical properties derived from a chimerical collagen found within their

primary structure domains that corresponds to collagen, polyhistidines and,

additionally, either elastin or dragline spider silk (Waite et al., 2003). These

threads undergo a 4-5-fold increase in tensile strength, due to aeration of

peptide-bonds 3, 4-dihydroxyphenylalanine (DOPA) and diDOPA by the

seawater (McDowell et al., 1999). Another example of intermolecular cross-

links and their influence on mechanical properties can be found in the

collagen of eggs of the sea urchin Strongylocentrotus purpuratus. The rapid

stabilization of the fertilization membrane by different cross-links makes it

refractory to chemical, enzymatic and mechanical disruption, thus creating a

protected environment for the embryo. The free radicals produced after

fertilization, and their subsequent exposure to seawater, contribute to

61

formation of di-tyrosine cross-links between the collagen molecules, thereby

hardening the egg-shell membrane (Foerder and Shapiro, 1977). The above

examples may also suggest that the amino acid sequence of S. ehrenbergi

collagen fibers, although still not known, determines their biomechanical

features (Fig. 17), thermal stability (Fig. 21), and water retentive capacity (Fig.

11) (see also ahead). In order to clarify this assumption, a proteomic study of

the amino acids sequence of the soft coral collagen peptides and the nature

of the different cross linking between them still needs to be conducted.

4.2 Light and electron microscopy

Both Masson Blue and Van Gieson stains homogenously stained the isolated

fibers (Fig. 7) and thus additionally confirmed their collagenous nature (Ross

and Wojciech, 2006). Fiber bundles of S. ehrenbergi revealed a packed

arrangement of almost round fibers, as seen in cross-section, coiled around

each other (Fig. 7). Such an arrangement is usually found in the connective

tissues of vertebrates such as tendons, ligaments and skin, where collagen

fibers are coupled with each other in an organized structure (Fratzl, 2008).

Furthermore, the soft coral single fibers featured a wavy course (Fig. 7a;

Fig.10d), and a parallel arrangement of fibrils, organized in a packed manner

(Fig. 8), which is typically found in fibers of fibrous tissue such as tendons

(Fratzl, 2008; Ushiki, 2002). Although the fiber bundles of the soft coral can

extend to a length which is comparable to the length of certain vertebrate’s

tendons and ligaments (Fratzl, 2003), elastin was not observed in the coral

fibers following Van Gieson staining (Ross and Wojciech, 2006) (Fig. 7c). This

is consistent with the published view that elastin is found exclusively in

61

vertebrates and not in invertebrates (Sage and Gray, 1980; Wagenseil and

Mecham, 2009). In addition, their diameter (9 ± 0.37 µm, n=166 fibers) is

significantly smaller than that of collagen fibers found in vertebrate connective

tissue (50-300 μm) (Fratzl, 2008). The soft coral fibrils are organized similarly

to collagen types I and III (Fratzl, 2008; Ottani et al., 2001). Fibrilar collagen

types I and III are the main constituents of the extracellular matrix of

metazoans and occur particularly in tissues exhibiting elasticity (Kuivaniemi et

al., 1997). A comparison between the structural features of the octocoral

fibers and its mesoglea (Fig 12i), in terms of fibril arrangement, indicated that

the arrangement of the former resembles tendons and ligaments rather than a

gelatinous mesoglea (Ottani et al., 2001). The dimensions and arrangement

of collagen fibers and fibrils in connective tissue determine their properties

and function. For example, the parallel alignment of fibers and fibrils in

tendons enhances their longitudinal strength, their random layered

organization in the skin maximizes compliance, the laminated layers in the

inter-vertebral discs provide flexibility, while the discontinuous fibers in the

highly hydrated matrix of the mesoglea lend extensibility and elasticity (Fratzl,

2008; Vogel, 2003). Therefore, it is suggested that the soft coral fibers feature

some functional similarities to tendons and ligaments, as derived from their

structural resemblance.

Although the fibrilar packing of the octocoral fibers revealed similarities to

collagen types I and III (Fig. 8), TEM micrographs of isolated coral fibrils by

sonication indicated that the diameter of the fibrils is 18.9 ± 2.4 nm (n=41)

(Fig. 9a) thus being more similar to type II fibrils (~ 35 nm) than to type I or III

62

fibrils (50-500 nm) (Antipova and Orgel, 2010; Fratzl, 2003; Kadler et al.,

1996; Ottani et al., 2002).

The collagen molecules in the fibril are arranged in a quasihexagonal lattice

lateral structure (Orgel et al., 2000), where each collagen molecule is offset

by ~30 nm with respect to its lateral neighbors. This gap is responsible for the

fibrils displaying alternating differences in electron density, with a 67 nm

repeat that corresponds to the gap and the overlap regions of the collagen

molecules (Toroian et al., 2007).

TEM images of negatively stained soft coral fibrils revealed an average

intensity distribution along the fibrils with major bands at 65-70 nm, and some

lower amplitude banding within the 65-70 nm unit (Fig. 9), corresponding to

published data on negatively stained collagens (Ortolani and Marchini, 1995).

There was a problem in assigning the precise D-period from high

magnification of TEM images, as there is little space for the stain to adhere

and it is impossible to mount the fibrils completely straight on one axis.

Nonetheless, the measurements provide an indication of the D-period of the

fibrils which was more precisely measured later by X-ray within the 65-70 nm

margin (see ahead).

In addition to the 65-70 nm bands, the negative staining revealed a parallel

arrangement of thinner sub-units organized in a packed manner within the

fibril (Fig. 9c). The width of these sub-units was ~2.5 nm, which corresponds

to single collagen molecules (Fratzl, 2008; Ottani et al., 2002). In comparison

63

with what is known for collagen molecule production and molecular structure,

very little is still understood regarding their three-dimensional arrangement in

the fibrils. Most models point to either a quasi-crystalline supramolecular

array of packed molecules, or to microfibrilar aggregates that interdigitate with

neighboring microfibrils to form the fibril, but no one model is universally

accepted (Orgel et al., 2006; Ottani et al., 2002; Perumal et al., 2008). The

dimensions of the sub-units noted within the soft coral fibers are assumed to

be external projections of the micro-fibrils or collagen molecules on the fibril

surface. This projection was further confirmed by TEM micrographs of a

collagen fibril protruding from a sectioned fiber, which showed uneven surface

area (Fig. 9d). The ability of TEM to reveal the complexity of the fibril surface

is not trivial, and may indicate an exceptionally rough fibril surface (Fratzl,

2008; Perumal et al., 2008). The surface of a fibril in fibrous collagens

comprises an interconnected area of collagen molecules and proteoglycans

(PG) that play an important role in restricting the fibrils’ growth and enable

their fusion (Fratzl, 2008). PGs were also noted by Cupromeronic Blue

staining (CB), which revealed a densely hydrated matrix, evenly distributed

between the fibrils and along the fibers (Fig. 10). PGs interact with collagen

through their globular protein cores, which are domains that form an N-

terminal hyaluronate-binding region that recognizes specific sequences in the

collagen composition (Fratzl, 2003; Roughley and Lee, 1994). The PG side

chains attract positively charged sodium ions (Na+), which attract water

molecules via osmosis (Martin et al., 2002; Movin et al., 1997). The PG and

the water create an extended and highly hydrated component in the fibrils,

which leads to the collagen fiber’s water retentive capacity and dictates some

64

of its biomechanical properties (Fratzl, 2003). For example, in the human

tendon, 40-70% of the dry weight is collagen and only 1% is a non-

collagenous extracellular matrix. However, this non-collagenous matrix

accounts for 65-75% of the total wet weight of a tendon due to the water

associated with the proteoglycans (Movin et al., 1997). The water and

proteoglycan matrix provides the lubrication and spacing that are crucial both

for the gliding function in fibrous collagen and in providing mechanical support

against compression (Sharma and Maffulli, 2006). The presence of PGs in

the soft coral collagen and its ability to strongly hold CB (noted by both the

naked eye and by TEM, Fig.8) is an indication of the water retentive capacity

of the studied fibers. This finding was also noted by wide-angle X-ray

diffraction (Fig. 11), which presented a significant background that covered

the first order, and indicated a water-rich fibrilar structure. This method also

confirmed the fibrilar nature of the soft coral collagen, its fibrilar packing

function, D- periodicity, helix pitch and helix molecular packing (see wide

angle X-ray diffraction results). The x-ray results also corresponded to the

TEM images, showing a similarity to the fibrilar packing revealed by the latter

(15.9± 3.11nm, n=15, Fig. 8) and by X-ray (16-17 nm). The similarity in

fibrilar packing between a rehydrated and mechanically sliced material (TEM),

and one that was not tampered with (X-ray), indicates a stable fibril

arrangement within the fiber.

The meridional 66 nm D-periodicity, derived from the X-ray studies, is shorter

than the known 67 nm periodicity of fibrilar collagen (Fratzl, 2008; Orgel et al.,

2006). Therefore, these results imply that the soft coral features a novel

65

collagen. The shorter D-periodicity may result either from a shorter

tropocollagen molecule, or a greater average molecular tilt relative to the fiber

axis than other known fibrilar types (Fratzel 2008). The helix pitch of the soft

coral collagen was approximately 2.86 angstroms (see X-ray result), thus

consistent with high proline content (Cameron et al., 2007). Both TEM and X-

ray diffraction indicated that the fibrilar soft coral collagen cannot be readily

identified as one of the previously known types. It possesses structural

features of both collagen types I, II and III, but it does not appear to be any of

these types, and its merdional 66 nm periodicity makes it difficult to

categorize among the known collagens. Although, the soft coral fibers display

a resemblance to tendons and ligaments in terms of fibril arrangement, their

water retentive capacity is more structurally related to the mesoglea. This

may indicate some kind of intermediate structural state between the

gelatinous mesoglea and fibrous collagen, as the ability to strongly retain

water is one of the properties that defines the differences between these two

types (Vogel, 2003). These findings support the hypothesis that the studied

fibers represent a new collagen, but there is still a need for a proteomic study

in order to verify this.

4.3 Location, distribution and formation of fibers within the colony

Histology revealed collagen fibers along six out of the eight mesenteries of the

polyps’ gastrovascular cavity. Although collagen is known to constitute a

major part in the cnidarian body wall (Barnes, 1994), there were differences

between the color intensity of the fiber bundles and that of the soft coral

mesoglea stained by Masson Blue (Figs. 10bc,12b). The binding of dyes to

66

collagen, which is a result of strong ionic linkages and hydrogen bonds to

amino groups causes connective tissues with different fibril concentrations

and packing to present different color intensity when subjected to a similar

staining protocol (i.e., exposure time and dye concentration) (Flint et al., 1975;

Ross and Wojciech, 2006). The soft coral fibers revealed higher color

intensity than the mesoglea (Figs. 10bc, 12b), thus indicating a different

arrangement and concentration of fibrils. These differences were clarified by

TEM images, which presented a linear packed fibril arrangement within the

fibers, and unorganized widely spaced fibrils in the mesoglea (Fig. 13a, 12i).

Bundles of coiled fibers within each mesentery (Fig. 12bc) were extended and

uncoiled after being pulled out from the tissue (Fig. 12def). The length of

isolated fibers, along with the histological sections (Fig. 12), indicates that

they are rather densely packed within the mesenteries (Fig. 12cf). SEM

images revealed that the fiber bundles stained by Masson Blue within the

mesenteries are composed of one or two fibers coiled together (Fig 10bce).

The arrangement of coiled long collagen fibers as seen in S. ehrenbergi

differs from the linear arrangement of collagen fibers in tendons, ligaments

and skin, or their embedment within a matrix, such as in bone and cartilage

(Kadler et al., 1996; Myllyharju and Kivirikko, 2001; van der Rest and

Garrone, 1991). The particular arrangement of collagen fibers in connective

tissues commonly indicates their function (Vogel, 2003). For example, tendon

fibers are linearly arranged and transmit force from one end to the other, while

in blood vessels collagen fibers create a reticulate structure that absorbs

elastic energy (Fratzl, 2008). Although the collagen fibers of S. ehrenbergi

67

possess unique biomechanical properties (see Results), examining their

internal arrangement within the coral mesenteries did not provide a clear

indication of their function in the tissue. To the best of my knowledge, there is

no previous record of tissues containing coiled, not stretched, collagen fibers

in metazoans. One indication of the functional role of these fibers is their

location within the mesenteries, which provides structural support for gonad

development, and the longitudinal retractor muscles (Fautin and Mariscal,

1991). In octocorals, 2 mesenteries are associated with the siphonoglyph, and

the other six with gonad development (Fautin and Mariscal, 1991; Galloway et

al., 2007). In the order Alcyonacea, gonads develop along the mesenteries,

and the developing gametogenic cells are mostly retained their until the

maturation process is completed (Benayahu and Loya, 1986; Simpson, 2009).

The coiled arrangement of the fibers along the free edge of the mesenteries

as fiber bundles that are enclosed in the tube-like structure of the

gastrodermis (Figs. 10 11a), may provide a supporting structure for the

mesenteries. The elastic coils inserted into the tubular structure can increase

its ability to endure horizontal forces and movements and prevent inward

collapse (Smith and Thomas, 1987). The fibers within the mesenteries may

similarly function, by utilizing their elastic properties (see results), and coiled

arrangement. This coiled arrangement may also support some elongation of

the mesenteries that is associated with gonad development (Fautin and

Mariscal, 1991; Galloway et al., 2007). This suggested model may provide an

explanation of the structural support, not only in the polyp mesenteries but

also for the whole colony, as collagen fibers were found along the stalk of the

colony (Fig. 15).

68

MRI images revealed collagen fibers extending from the polyps to the

periphery of the stalk down to the base (Fig. 16). Histological sections of the

stalk further supported the MRI results and indicated that the fibers are

located in mesentery-like structures of the gastrodermal cavities that extend

into the basal part of the stalk (Fig. 15). These gastrodermal cavities are

denser in the stalk periphery than at its center. Octocorals, including

Sarcophyton spp., contain solenia lined with gastrodermal cells within their

coenenchyme, forming a network of gastrodermal canals interconnecting the

gastric cavities of polyps (Bayer et al., 1983; Fabricius and Alderslade, 2001).

Although the gastrodermal canals are considered as the graduall narrowing of

the basal part of the polyps gastric cavity (Bayer et al., 1983; Fabricius and

Alderslade, 2001), there are no published data relating to their containing a

mesentery-like structure, as found in the stalk of S. Ehrenberg (Bayer et al.,

1983; Fabricius and Alderslade, 2001; Fautin and Mariscal, 1991; Galloway et

al., 2007). Small gastrodermal cavities are concentrated at the periphery of

the stalk and their fiber-bearing mesenteries make this area of the stalk more

dense with fibers than the central part of the stalk. This may indicate that the

fibers function as reinforcing structures. Interestingly, in blood vessels,

collagen fibers create such reinforcing structures based on external/peripheral

support (Holzapfel, 2001), and the same fiber-based external support can be

found in plants such as the bamboo (Nogata and Takahashi, 1995). One

might similarly look at the stalk of S. ehrenbergi, as a cylindrical column with

a peripheral support of collagen fibers. In order to verify this structural

assumption, however, there is a need for a comparative biomechanical study

69

of S. ehrenbergi’s different colony parts. The results of such study should be

compared to those of other congenerics that present some of S. ehrenbergi’s

morphological features, such as S. glaucum, which was partly studied by

Koehl (1982), and shares the same habitat but does not possess these long

fibers in its tissue.

In addition to the fibers within the mesenteries, histology and TEM revealed

laminated collagenous fibers within the mesoglea around the sites of

decalcified sclerites (Fig. 14f). These resemble sheaths comprised of distinct

layered fibers, differing from both the mesogleal scattered fibrils (Fig.12g-i)

and the mesenterial fibers (Fig.11a). Both these laminated fibers and the

mesenterial ones are associated with the striated vesicles located within

certain gastrodermal cells of the mesenteries (Fig.11a-c) and within cells

adjacent to the sclerite sites (Fig. 14a, b). Although the structural appearance

of these vesicles are similar in both locations, the cells of the mesenteries are

considered of endodermal origin, while those residing within the mesoglea are

of ectodermal origin (Fautin and Mariscal, 1991). The development of the fine

fibrils and their association with collagen fibers and connective tissue in

metazoans is still far from being fully understood (Fratzl, 2008). The process

by which molecules in the cell are transported from their site of synthesis in

the endoplamic reticulum through the Golgi complex has long been studied

(Bonifacino and Glick, 2004). Vesicles are abundant in the vicinity of the Golgi

complex, and a wealth of evidence favors a model in which cargo is

transported by incorporation into vesicles and budding from the membrane

(Bonifacino and Glick, 2004). There is controversy as to whether the vesicular

71

transport model is generally applicable to the biosynthesis of collagen, since

the vesicles studied so far were only 60-80 nm in diameter, whereas the triple

helical domains of the fibril-forming collagens are ~300 nm long (Canty and

Kadler, 2005). The location, relatively large size (>500 nm, Fig.11 -12), and

the striated appearance of the soft coral vesicles, may support the vesicular

transport model regarding collagen fibrillogenesis in S. ehenbergi. Future

studies should further investigate the interaction between the fibrous

assemblages found in the studied octocoral and the cells surrounding them.

This should include molecular and proteomic studies of the vesicles in order

to fully understand their structure and function in conjunction with the

fibrillogenesis process.

4.4 Biomechanical and physical properties of the fibers

The collagen fibers of S. ehrenbergi featured an impressive stretching ability,

to a high strain (19.4±4.27%, n=12), without failing or undergoing irreversible

damage. In contrast, mammalian collagen fibers can be reversibly stretched

to strains of only 8-10% without failure (Danto and Woo, 1993; Fung and Liu,

1995; Vogel, 2003). The mesoglea, on the other hand, as a non- organized

gelatinous structure comprised of thin and short fibrils, can stretch to strains of

350-600% (Koehl, 1982). The stiffness of the soft coral fibers (0.44 ± 0.1 GPa,

n =12) is about half to one-third lower than the reported range for mammalian

fibers (0.9–1.8 GPa) (Sverdlik and Lanir, 2002), and five orders of magnitude

higher than for the mesoglea (0.01 MPa) (Vogel, 2003). Their average stress

to failure (49.4 ± 11.7 MPa, n=12) is about one-half the reported tensile

strength for mammalian collagen fibers (100 MPa) (Vogel, 2003) and one

71

order of magnitude higher than for the mesoglea (1-2.5 MPa) (Koehl, 1982).

The soft coral fiber’s strain-stress curves of preconditioning cycles (Fig. 17)

revealed a typical tendon and ligament behavior, with a noted difference

between L initial and L0 (Vogel, 2003). The difference between L initial and L0

resulted from the fibrils’ arrangement within the fiber (Lokshin and Lanir,

2009). When the fiber is stretched for the first time, the fibrils are getting

organized and in lines along it, resulting in a different stress-strain curve than

the following cycles, which are similar to each other. This biomechanical

behavior is characteristic of collagens with a tight-fitting arrangement, such

as tendons, where the fibrils are long relative to their diameter (Provenzano

and Vanderby, 2006), and the shear transfer between them through the matrix

is sufficient to allow the fibers to behave like continuous fiber composite

material (Fratzl, 2008; Ker, 2007). This biomechanical property is not

displayed in the mesoglea, which consists of short fibrils surrounded by a

thick and soft matrix, and which can deform to an extension of several

hundred percent (large strains) through the sliding of its fibrils relative to their

neighbors (Alexander, 1962; Koehl, 1977).

Table 2. The soft coral Sarcophyton ehrenbergi. Summarize of material properties in

comparison to other known collagens (sources: Koehl, 1982; Sverdlik and Lanir, 2002; Vogel,

2003).

Material

Extensibility (%)

Modulus of elasticity -stiffness (GPa)

Tensile strength (MPa)

Mammalian tendons and ligaments 8-10 0.9-1.8 100

S. ehrenbergi fibers 19.4±4.27 0.44±0.1 49.4±11.7

Mesoglea 350-600 0.01 1-2.5

Mussel byssus threads 100 0.1 50

Spider silk 30-1600 10 2000

72

The in situ SPM-TEM mechanical characterization (Figs. 16-17) of the current

study revealed a strong fibril association/connection within the soft coral

fibers. The recorded forces of detachment from the fibers exceeded 8-9 µN,

which was the limit of the experimental setup capabilities. These findings,

along with the light microscopy, SEM TEM, MRI and X-ray studies, thus

support the assumption that the soft coral fibers present an internal

organization that strikingly resembles vertebrate tendons yet differs from the

cnidarian collagenous mesoglea. Given this structural resemblance, the

differences in mechanical properties between the soft coral fibers and

vertebrate tendons are probably derived almost entirely from the composite-

like organization of the former (Vogel, 2003). These differences can be

attributed to the collagen fibrils, their matrix or both (Vogel, 2003). The

current study revealed differences between the soft coral collagen and the

collagen types I-III in peptide components, fibril structure and fibril

arrangement, thereby indicating that these structural features influence the

mechanical properties of the soft coral collagen. However, the water retentive

capacity of the fibers and their hydrated proteoglycan matrix (Figs. 8, 9)

suggest that the matrix surrounding the soft coral collagen fibrils also

contributes to their mechanical properties.

S. ehrenbergi is a benthic organism found in habitats exposed to strong tidal

currents and typhoons (Dai, 1993). It is suggested that colonies of this

species utilize their collagen fibers to form a structural support that will

withstand the strong hydro-mechanical forces in their environment. Similarly,

the sea anemone Metridum senile withstands drag forces by utilizing its thick

73

jelly-like mesoglea for stretching several-folds under harsh conditions

(Gosline, 1971). The elastic capabilities of S. ehrenbergi’s fibers, and their

higher abundance in the periphery of its stalk, also support such a conclusion.

Future studies should deal with those congeners which inhabit other reef

habitats, in relation to the properties of their collagen fibers.

As all of the biomechanical tests in this study was done on fibers that were

stored in 70% ethanol before rehydration, it is recommended that farther

studies should test freshly isolated fibers in order eliminate any preservation

related bias.

The thermogravimetric analysis (TGA) and differential scanning calorimetry

provided additional evidence for the water retentive capacity of the soft coral

fibers revealed by TEM and X-ray (Figs. 8, 9). TGA of stored and dried fibers

showed a 7-30% weight loss when they were heated to ~1000C (Fig. 20). This

loss resulted from the evaporation of the solvents and indicated the ability of

the fibers to strongly hold the solvents (Artiaga et al., 2005; Golodnitsky et al.,

2003). Evaporation is a process that requires energy (heat consumption) and

therefore could mask the enthalpy point (Artiaga et al., 2005). Although

vacuum-dried fibers still showed solvent evaporation (~7%), they were used

for the DSC analysis in order to minimize the masking effect. The soft coral

collagen exhibits an unexpectedly high denaturation temperature of

67.8°C(Fig. 21), in contrast to native collagen types I and II, which

demonstrate a value of only 42°C for soluble tropocollagen. The latter

increase to ~54°C upon fibrillation and reach 67°C only when artificially cross-

linked (Miles et al., 1995; Tiktopulo and Kajava, 1998). The thermal stability of

74

collagens predominantly depends on the formation of intermolecular lysyl

oxidase-derived cross-links (Fratzl, 2008). The high denaturation point of S.

ehrenbergi fibers implies that its collagen fibers are naturally cross-linked.

However, biochemical and chemical studies are still needed in order to

validate this assumption.

4.5 Summary

Prior to the current study there was no evidence among invertebrates for the

presence of internal long collagenous fibers that can be observed by the

naked eye (Aouacheria et al., 2006; Bell and Gosline, 1996; Elder, 1973; Har-

el and Tanzer, 1993; Helman et al., 2008; Tucker et al., 2011). The finding of

tendon-like, long collagen fibers in the soft coral S. ehrenbergi which is a two

cell-layered organism, has altered our knowledge on the appearance and

diversity of collagen in metazoans. Throughout evolution the diversity of

fibrilar collagen chains has increased, as well as the different forms of their

maturation and interactions (Exposito et al., 2002). The collagen fibers of S.

ehrenbergi present a mixed set of properties, some of which resemble those

in vertebrates or invertebrates, while others are novel. A better understanding

of the molecular structure of this collagen, along with genomic studies of the

cells revealed to be associated with it, may contribute to our understanding of

the evolution of collagen and its fibrillogenesis. Elucidation of the molecular

structure of this collagen and its physical properties will conduce to assessing

the feasibility of using it for biomedical applications. Collagen provides

biomaterial for a myriad of uses, and is extensively utilized in tissue

engineering as scaffolding for repair or augmentation of body tissue

(Gentleman et al., 2003; Lee et al., 2001). The collagen fibers of S.

75

ehrenbergi present a suite of properties, such as thermal stability, fibrilar

organization, biomechanical and water retentive capacities that could make

them suitable for biomedical applications. In order to explore such

possibilities, in situ and in vitro studies on possible interactions between the

fibers and vertebrate cells (murine) have already commenced (Benayahu et

al., 2011).

Preliminary findings also indicate that the fibers may provide to taxonomic

identification of a cryptic taxon of alcyoniid octocorals, comprised of a mix of

nominal species from the genera Sarcophyton and Lobophytum. (McFadden

et al., 2006). Species of these soft corals genera , have a wide Indo–Pacific

distribution (Fabricius and Alderslade, 2001), can be found on the reefs in

similar ecological niches, and certain species reveal close morphological

features. Interestingly, some species of this suspected cryptic taxon present

fibers within their tissue (preliminary work, Sella& Benayahu). This may

provide morphological features which support the findings of the molecular

study and can help taxonomic identification.

It is expected that the current results, which were initiated as field

observations and continued in laboratory studies, will further advance our

knowledge on Octocorallia taxonomy, collagen among lower invertebrates, its

fibrillogenesis, and its potential for practical biomedical applications.

76

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תקציר

שלהם , רדיאלית ומופע מדוזה או פוליפ-מערכת הצורבנים כוללת בעלי חיים בעלי סימטריה רדיאלית או בי

על ידי שכבה לא המופרדים ,אנדודרם ואקטודרם דו שכבתיים בעליהם . חלל בעל פתח מוקף זרועות צייד

המצופה ופיינת בלוע מחלקת האלמוגים כוללת בעיקר בעלי חיים ישיבים ומא. תאית המכונה מזוגליאה

, מינים לערך 3,211כוללת ה ,השמונאים תמחלק-תתמצויה מחלקה זוב. ומחיצות גוף רדיאליות םאקטודרב

השמונאים הם בעלי חיים מושבתים בעלי . Helioporace ו , Alcyonacea, Pennatulaceaמהמחלקות

. מחטי שלד ותעלות גסטרודרמאליות, ומורכב בעיקר ממזוגליאה, coenenchymeהמכונה תווך בין רקמתי

המזוגליאה יחסית מפותחת באלמוגים . פוליפ השמונאים מאופיין בשמונה מחיצות ושמונה זרועות צייד מנוצות

תכונות קרובות למטריקס שלה ,עיקר תפקידה של המזוגלאה. ממשקלם הכוללניכר לעתים חלק ומהווה , אלו

. סיבי שרירבתוכה מספקת תמיכה לשלד ההידרוסטטי של המושבה ומעגנת היא . הוא מכאני, חוץ תאי

קולגן הוא אחד . ממטריקס עשיר במים עשוי רב סוכרים ופרוטוגליקנים ומערכת סיבי קולגן תמורכבמזוגליאה ה

למבנים סיבים ד לטין וע'הוא מאורגן באופן היררכי ומציג מבנים שונים מג, החלבונים הנפוצים בבעלי חיים

אשר נכרכות אחת על השנייה ,כל קבוצות הקולגן מורכבות משלוש שרשראות חלבוניות. כגידים ורצועות

יוצרות היוצרות את המיקרופבירילות והפיברילות , מולקולות אלו. המכונה טרופוקולגן ,ליצירת סופרהליקס

ה בהרכב חומצות האמינו בשרשראות קבוצות הקולגן השונות נבדלות אחת מהשני. את סיב הקולגן

.שיוצרות את מולקולת הטרופוקולגן ,ובהרכב שלוש השרשראות השונות, הפוליפפטיד

Sarcophyton ehrenbergiשנמצאו באלמוג השמונאי ,סיביםההייתה לחקור את טיבם של הזמחקר מטרת

(Alcyoniidae) .ונמצא בסימביוזה עם אצות חד תאיות ,סוףובכללם ים , אלמוג זה חי באזורי ריף שונים בעולם

zooxanthellae .עשרות סנטימטר ובקוטר של עד ת האלמוג חושפת צרורות סיבים באורך ורקמה של קריע

Nuclear magnetic resonance analysesבדיקת . של מאות מיקרון הניתנים למשיכה מתוך הרקמה

, פרולין, מצות אמינו זיהתה ריכוזים גבוהים של גליציןסיבים ואנליזת חושל החלבוני החשפה את טבעם

המחקר עסק בתכונות . את השערת המחקר לגבי הרכב הסיביםאוששה המאפיינים קולגן ו ,והידרוקסיפרולין

לצורך . מיקומם במושבההוא חשף את ובנוסף , של סיבי הקולגן של האלמוג תוביו מכאניומבניות , ביוכימיות

מיקרוסקופית . מבחנים ביומכאנים ואנליזת חומרים, הדמיה מגנטית, ותבשיטות מיקרוסקופינעשה שימוש , כך

סיבי האלמוג מציגים . בעם הקולגנייאת טו גם הם אור וצביעות היסטולוגיות של סיבי אלמוג מבודדים אישר

נמצא (µm, n=166 0.37 ± 9)למרות שקוטר הסיבים. הנכרכים אחד על השני ,מבנה כמעט עגול של סיבים

סידור הרי , (μm 50-300) ברקמת חיבור של חולייתניםאשר מצויים ,קטן משמעותית מקוטר סיבי קולגן

תת יחידות מאורגנות וצפופות נמצאו ,בנוסף. I-IIIאותם זהה לקבוצות קולגן יםהמרכיב ,הסיבים והפיברליות

ספציפית הצביע. (nm wide 2.5~)קולגן או מיקרופיברילה המולקולת את על פני הפיברילות בגודל התואם

ממצא , מטריקס פרוטוגליקני עשיר במים הראתה( Cupromeronic Blue)מקרוסקופיה אלקטרונית חודרת ל

שהתגלו ממצאים נוספים . שזיהתה מבנה פיברלי עשיר במים ,Xקרינת תוצאות דיפרקציית גם על ידי נתמךה

. helix molecular packing ו fibrilar packing function ,D- periodicity ,helix pitchבדיפרקציה היו

כאשר סיבים .על יכולת אחיזת המים של הסיביםגם הם הצביעו ,Thermogravimetric analysisממצאי

י למרות הדמיון המבני בין סיב. C o111-לבחימום 7-31% שיובשו בשיטות שונות הראו איבוד מים של

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Differential scanningאנליזת . המסודר ברקמות חיבור של חולייתנים ,לטנית והקולגן הסיבי'הג

calorimetry כי טמפרטורת ההתכה של סיבי האלמוג גבוהה במיוחד ,הראתה (67.8°C ) וזהה לזו של קולגן

הראו תוצאות המחקר הביומאכני של סיבי אלמוג מבודדים . תלאכותימ (cross-linking) קשירה צולבת יאחר

יכולת מתיחה כמו ,מאשר לאלו של מזוגליאה ,ורצועותאשר קרובות יותר לאלו של גידים ,סדרת תכונות

. MPa 11.7 ± 49.4 ועומס קריעה, GPa 0.1 ± 0.44קשיות , 19.4±4.27%

ופיזורם הסיבים םאת מיקו הראתה, הדמיה מגנטית של מושבה שלמה ומיקרוסקופיה של חלקיה השונים

ארוזים לאורך קצה המחיצה ה ,סיבי קולגן שש מתוך שמונה מחיצות הפוליפ מכילות. ברקמת האלמוג

מגיעות עד לבסיס ה ,בגזע נמצאים הסיבים במחיצות של תעלות גסטרודראמליות. ונמשכים לגזע המושבה

-תפקיד מבנילהם ניתן להניח ש, מתוך מיקום הסיבים במושבת האלמוג ותכונותיהם הביומכאניות. המושבה

,המורכב מסיביםנוסף ברקמת האלמוג מבנה נמצאך המחקר במהל. רמת הפוליפ והמושבה כולהב תומך

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,גודלם. סיביהנראה , בעלות תוכןוויסקולות , ןקולגהסמוכים לבתאי רקמה נמצאו בשני המקרים . המחיצות

.תהליך יצירת סיבי הקולגן באלמוגב יתכן והן משתתפותכי , מרמזמיקומם ומראם

.Sאלו שנמצאים באלמוג השמונאי בדומה ל ארוכיםפנימיים וסיבי קולגן עד כה לא תועדו בחסרי חוליות

ehrenbergi . ההופעההסתכלות על הבעל חיים דו שכבתי משנה את ת ברקמ מציאת סיבים דמויי גידים

, חלקן קרובות לחולייתנים, מציגים מגוון תכונות הרךסיבי הקולגן של האלמוג . החיעולם והתפתחות של קולגן ב

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אשר , ביומכאניות ותאחיזת מים יכולות, מבנה סידורי, מציגים סדרת תכונות כדוגמת טמפרטורת התכה גבוהה

לנוכחות ש ,מראותאף וצאות ראשונית ת. ןושחזוררקמות יכולות לעשותם מתאימים לשימושים כגון הנדסת

שהוצע שהיא מהווה , Alcyoniidae -לקבוצת מינים ממשפחת המורפולוגי -טקסונומיכסמן סיבים גם ערך ה

השילוב בין ממצאי וידגיש את מגוונים אשר יקדם תחומי מחקר ,הווה בסיס ידעמהמחקר הנוכחי . סוג קריפטי

. כאחד יישומיבסיסי וקר מחמחקר במעבדה למטרות ועבודת שדה

עבודה זו נעשתה בהדרכת

יהודה בניהו' פרופ

אביב -אוניברסיטת תל וייז. ס' ורג'ש ג"הפקולטה למדעי החיים ע

המחלקה לזואולוגיה

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Sarcophyton ehrenbergi הרך בצקנית

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