Text - SeArc

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
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
i
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 microfibrils 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
ii
confirmed the fibrilar nature of the soft coral collagen, its fibrilar packing function, Dperiodicity, 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.
iii
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.
iv
v
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
1
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
2
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
3
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
4
(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).
5
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 unstretched 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
6
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
7
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 extracellular 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
8
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 righthanded 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)
9
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).
11
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 twoelement system enables the connective tissues to tolerate both pulling and
12
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,
13
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).
14
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
15
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 stalkcircumference 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
16
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).
17
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
18
physical properties of the coral collagen fibers is also important in order to
assess the feasibility of utilizing them for biomedical applications.
19
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 seawatersystem. The system comprised five PVC tanks, 1 m 3 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 coralsand (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
21
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.
22
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
23
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 bluemucopolysaccharides 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
24
fibrils in TEM micrographs. This was done in order to locate repeated staining
patterns along the fibrils. The color intensity histogram displayed a twodimensional 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 Raxis 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).
25
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 fatsuppressed 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
26
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
ba
calculated beam deflection was subtracted from all fiber displacement results
in order to obtain the real displacement. In the experiments, undulated fiber
27
was extended until becoming straight but not stretched, and its reference
length was measured by caliper in mm (L initial). In order to locate L 0, 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.
28
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 TEMSPM 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 semicircular 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).
29
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 512 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 nonreversing 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).
31
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).
32
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.
33
Figure 6. The soft coral Sarcophyton ehrenbergi. Amino acid analysis of fibers featuring
collagen-related amino acids. Glycine, Proline, and Hydroxyproline are indicated.
34
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.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Sum
Name
Hpro
AMQ
Asp
Ser
Glu
Gly
His
NH3
Arg
Thr
Ala
Pro
Tyr
Val
Met
Lys
lle
Leu
Phe
RT
11.797
12.608
13.625
15.347
16.013
17.494
18.262
20.05
21.306
21.666
21.975
22.815
23.115
24.387
25.043
26.748
27.852
28.356
29.306
29.788
30.378
31.133
31.867
32.46
32.884
33.72
34.587
Area
1449440
210420
2201385
1465071
2789428
7795628
465666
4225440
80331
2435609
1571415
56598
3024957
28980
1105715
22910
60417
743866
3534428
1366922
52533
25901
1399282
2794982
3700211
2419376
85143
Height
98789
10577
143512
97687
177478
423228
26694
221045
7356
222943
126517
7125
238777
1865
101985
2261
7533
91521
356606
147856
5555
1890
158919
294791
392968
260384
7256
Amount (pmol)
83.674
43.312
168.082
87.668
191.959
501.615
18.943
149.948
% from known AA
4.15
2.15
8.34
4.35
9.52
24.89
0.94
7.44
107.777
69.217
5.35
3.43
130.905
6.49
102.471
5.08
30.726
79.999
39.482
1.52
3.97
1.96
59.693
49.841
66.24
33.978
2.96
2.47
3.29
1.69
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 ropelike 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 acellular, 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
35
collagenous nature (Fig. 7a, b, c). The blue (Masson Blue) and the red (Van
Gieson) stains revealed the same degree of coloration on all longitudinallysectioned 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).
36
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.
37
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
showed dark and repeated
intensity distribution,
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
38
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.
39
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).
41
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.
42
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 tubelike 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
43
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.
44
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).
45
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.
46
Figure 14. The soft coral Sarcophyton ehrenbergi. Microscopy of Mesoglea. a Longitudinalsections 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.
47
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.
48
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).
49
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 loadingunloading), 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.
51
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.
52
Fibril- Fiber Desplacment
5
4
Force (µN)
3
2
1
0
-1
-2
0
20
40
60
80
100
120
140
160
180
200
Distance (nm)
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.
53
Figure 19. The soft coral Sarcophyton ehrenbergi. Mechanical characterization of nonisolated 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.
54
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.
55
120
–––––––
––––
––––– ·
collagen vac dried tubes.001
Collagen-1 24h R-airTGA.001
Collagen-1-TGA.001
115.96°C
100
7.383%
(0.1689mg)
Weight (%)
tubes dried in vacuum.
12.95%
(0.2464mg) tubes dried 24h at RT on filter pap in air atmos.
80
30.40%
(1.723mg)
tubes as is
60
40
0
50
100
150
200
Temperature (°C)
250
300
350
Universal V2.6D TA Instruments
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,
0
25 C (green). Note that even vacuum-dried fibers showed 7.383% weight loss when heated
0
to ~100 C.
56
Figure 21. The soft coral Sarcophyton ehrenbergi. Differential scanning calorimetry (DSC) of
0
isolated collagen fibers. a Two heat consumption areas along heating profile: at 42-54 C and
0
0
at 61-74 C (marked). Denaturation occurred at ~ 67.8°C (arrow). b Detailed DSC of 42-54 C
heat consumption areas that is rejected as the denaturation area; note the gradual change in
heat flow, and the lack of distinctive peak.
57
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
58
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 crosslinks 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
59
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
61
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
62
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 Nterminal 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
63
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 noncollagenous 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
64
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 Xray 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
65
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
66
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).
67
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
68
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
69
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
Mussel byssus threads
350-600
100
0.01
0.1
1-2.5
50
Spider silk
30-1600
10
2000
71
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 compositelike 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
72
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 crosslinked (Miles et al., 1995; Tiktopulo and Kajava, 1998). The thermal stability of
73
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; Harel 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.
74
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.
75
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83
‫תקציר‬
‫מערכת הצורבנים כוללת בעלי חיים בעלי סימטריה רדיאלית או בי‪-‬רדיאלית ומופע מדוזה או פוליפ‪ ,‬שלהם‬
‫חלל בעל פתח מוקף זרועות צייד‪ .‬הם דו שכבתיים בעלי אנדודרם ואקטודרם‪ ,‬המופרדים על ידי שכבה לא‬
‫תאית המכונה מזוגליאה‪ .‬מחלקת האלמוגים כוללת בעיקר בעלי חיים ישיבים ומאופיינת בלוע המצופה‬
‫באקטודרם ומחיצות גוף רדיאליות‪ .‬במחלקה זו מצויה תת‪-‬מחלקת השמונאים‪ ,‬הכוללת ‪ 3,211‬מינים לערך‪,‬‬
‫מהמחלקות‬
‫‪ ,Alcyonacea, Pennatulacea‬ו ‪ . Helioporace‬השמונאים הם בעלי חיים מושבתים בעלי‬
‫תווך בין רקמתי המכונה ‪ ,coenenchyme‬ומורכב בעיקר ממזוגליאה‪ ,‬מחטי שלד ותעלות גסטרודרמאליות‪.‬‬
‫פוליפ השמונאים מאופיין בשמונה מחיצות ושמונה זרועות צייד מנוצות‪ .‬המזוגליאה יחסית מפותחת באלמוגים‬
‫אלו‪ ,‬ומהווה לעתים חלק ניכר ממשקלם הכולל‪ .‬עיקר תפקידה של המזוגלאה‪ ,‬שלה תכונות קרובות למטריקס‬
‫חוץ תאי‪ ,‬הוא מכאני‪ .‬היא מספקת תמיכה לשלד ההידרוסטטי של המושבה ומעגנת בתוכה סיבי שריר‪.‬‬
‫המזוגליאה מורכבת ממטריקס עשיר במים עשוי רב סוכרים ופרוטוגליקנים ומערכת סיבי קולגן‪ .‬קולגן הוא אחד‬
‫החלבונים הנפוצים בבעלי חיים‪ ,‬הוא מאורגן באופן היררכי ומציג מבנים שונים מג'לטין ועד למבנים סיבים‬
‫כגידים ורצועות‪ .‬כל קבוצות הקולגן מורכבות משלוש שרשראות חלבוניות‪ ,‬אשר נכרכות אחת על השנייה‬
‫ליצירת סופרהליקס‪ ,‬המכונה טרופוקולגן‪ .‬מולקולות אלו‪ ,‬יוצרות את המיקרופבירילות והפיברילות היוצרות‬
‫את סיב הקולגן‪.‬‬
‫קבוצות הקולגן השונות נבדלות אחת מהשניה בהרכב חומצות האמינו בשרשראות‬
‫הפוליפפטיד‪ ,‬ובהרכב שלוש השרשראות השונות‪ ,‬שיוצרות את מולקולת הטרופוקולגן‪.‬‬
‫מטרת מחקר זה הייתה לחקור את טיבם של הסיבים‪ ,‬שנמצאו באלמוג השמונאי ‪Sarcophyton ehrenbergi‬‬
‫)‪ .(Alcyoniidae‬אלמוג זה חי באזורי ריף שונים בעולם‪ ,‬ובכללם ים סוף‪ ,‬ונמצא בסימביוזה עם אצות חד תאיות‬
‫‪ .zooxanthellae‬קריעה של רקמות האלמוג חושפת צרורות סיבים באורך של עד עשרות סנטימטר ובקוטר‬
‫של מאות מיקרון הניתנים למשיכה מתוך הרקמה‪ .‬בדיקת ‪Nuclear magnetic resonance analyses‬‬
‫חשפה את טבעם החלבוני של הסיבים ואנליזת חומצות אמינו זיהתה ריכוזים גבוהים של גליצין‪ ,‬פרולין‪,‬‬
‫והידרוקסיפרולין‪ ,‬המאפיינים קולגן ואוששה את השערת המחקר לגבי הרכב הסיבים‪ .‬המחקר עסק בתכונות‬
‫ביוכימיות‪ ,‬מבניות וביו מכאניות של סיבי הקולגן של האלמוג‪ ,‬ובנוסף הוא חשף את מיקומם במושבה‪ .‬לצורך‬
‫כך‪ ,‬נעשה שימוש בשיטות מיקרוסקופיות‪ ,‬הדמיה מגנטית‪ ,‬מבחנים ביומכאנים ואנליזת חומרים‪ .‬מיקרוסקופית‬
‫אור וצביעות היסטולוגיות של סיבי אלמוג מבודדים אישרו גם הם את טיבעם הקולגני‪ .‬סיבי האלמוג מציגים‬
‫מבנה כמעט עגול של סיבים‪ ,‬הנכרכים אחד על השני‪ .‬למרות שקוטר הסיבים )‪ (9 ± 0.37 µm, n=166‬נמצא‬
‫קטן משמעותית מקוטר סיבי קולגן‪ ,‬אשר מצויים ברקמת חיבור של חולייתנים )‪ ,(50-300 μm‬הרי סידור‬
‫הסיבים והפיברליות‪ ,‬המרכיבים אותם זהה לקבוצות קולגן ‪ .III-I‬בנוסף‪ ,‬נמצאו תת יחידות מאורגנות וצפופות‬
‫על פני הפיברילות בגודל התואם את מולקולת הקולגן או מיקרופיברילה )‪ .(~2.5 nm wide‬צביעה ספציפית‬
‫למקרוסקופיה אלקטרונית חודרת (‪ )Cupromeronic Blue‬הראתה מטריקס פרוטוגליקני עשיר במים‪ ,‬ממצא‬
‫הנתמך גם על ידי תוצאות דיפרקציית קרינת ‪ ,X‬שזיהתה מבנה פיברלי עשיר במים‪ .‬ממצאים נוספים שהתגלו‬
‫בדיפרקציה היו ‪ helix pitch ,D- periodicity , fibrilar packing function‬ו ‪. helix molecular packing‬‬
‫ממצאי ‪ ,Thermogravimetric analysis‬הצביעו גם הם על יכולת אחיזת המים של הסיבים‪ .‬כאשר סיבים‬
‫שיובשו בשיטות שונות הראו איבוד מים של ‪ 7-31%‬בחימום ל‪ .111o C -‬למרות הדמיון המבני בין סיבי‬
‫האלמוג וגידים ורצועות של חולייתנים‪ ,‬יכולות אחיזת המים המשמעותית של סיבי האלמוג קרובה יותר‬
‫לתכונות המזוגליאה‪ .‬יתכן‪ ,‬שממצאים אלו מצביעים על כך‪ ,‬שסיבי האלמוג מהווים מצב ביניים בין המזוגליאה‬
‫הג'לטנית‬
‫והקולגן הסיבי‪ ,‬המסודר ברקמות חיבור של חולייתנים‪ .‬אנליזת‬
‫‪Differential scanning‬‬
‫‪ calorimetry‬הראתה‪ ,‬כי טמפרטורת ההתכה של סיבי האלמוג גבוהה במיוחד ( ‪ )67.8°C‬וזהה לזו של קולגן‬
‫אחרי קשירה צולבת ( ‪ )cross-linking‬מלאכותית‪ .‬תוצאות המחקר הביומאכני של סיבי אלמוג מבודדים הראו‬
‫סדרת תכונות‪ ,‬אשר קרובות יותר לאלו של גידים ורצועות‪ ,‬מאשר לאלו של מזוגליאה‪ ,‬כמו יכולת מתיחה‬
‫‪ ,19.4±4.27%‬קשיות ‪ ,0.44 ± 0.1 GPa‬ועומס קריעה ‪.49.4 ± 11.7 MPa‬‬
‫הדמיה מגנטית של מושבה שלמה ומיקרוסקופיה של חלקיה השונים‪ ,‬הראתה את מיקום הסיבים ופיזורם‬
‫ברקמת האלמוג‪ .‬שש מתוך שמונה מחיצות הפוליפ מכילות סיבי קולגן‪ ,‬הארוזים לאורך קצה המחיצה‬
‫ונמשכים לגזע המושבה‪ .‬בגזע נמצאים הסיבים במחיצות של תעלות גסטרודראמליות‪ ,‬המגיעות עד לבסיס‬
‫המושבה‪ .‬מתוך מיקום הסיבים במושבת האלמוג ותכונותיהם הביומכאניות‪ ,‬ניתן להניח שלהם תפקיד מבני‪-‬‬
‫תומך ברמת הפוליפ והמושבה כולה‪ .‬במהלך המחקר נמצא ברקמת האלמוג מבנה נוסף המורכב מסיבים‪,‬‬
‫אשר עוטף את מחטי השלד‪ ,‬כפי שאותר לאחר המסתם‪ .‬מבנה סיבי זה שונה מהסיבים הארוכים שלאורך‬
‫המחיצות‪ .‬בשני המקרים נמצאו בתאי רקמה הסמוכים לקולגן‪ ,‬וויסקולות בעלות תוכן‪ ,‬הנראה סיבי‪ .‬גודלם‪,‬‬
‫מיקומם ומראם מרמז‪ ,‬כי יתכן והן משתתפות בתהליך יצירת סיבי הקולגן באלמוג‪.‬‬
‫עד כה לא תועדו בחסרי חוליות סיבי קולגן פנימיים וארוכים בדומה לאלו שנמצאים באלמוג השמונאי‬
‫‪S.‬‬
‫‪ .ehrenbergi‬מציאת סיבים דמויי גידים ברקמת בעל חיים דו שכבתי משנה את ההסתכלות על ההופעה‬
‫והתפתחות של קולגן בעולם החי‪ .‬סיבי הקולגן של האלמוג הרך מציגים מגוון תכונות‪ ,‬חלקן קרובות לחולייתנים‪,‬‬
‫חלקן לחסרי חוליות וחלקן חדשות‪ ,‬שטרם תוארו‪ .‬הבנה טובה יותר של מבנהו המולקולרי של החלבון ומחקר‬
‫גנומי של תאים שנמצאים בקרבת סיבים אלו יכולה לקדם את ההבנה של תהליכי יצירת הקולגן גם בהיבטים‬
‫אבולוציוניים‪ .‬בנוסף‪ ,‬הבנה זו תתרום להערכת יכולתו של קולגן זה לשמש ליישומים רפואיים‪ .‬סיבי האלמוג‬
‫מציגים סדרת תכונות כדוגמת טמפרטורת התכה גבוהה‪ ,‬מבנה סידורי‪ ,‬יכולות ביומכאניות ותאחיזת מים‪ ,‬אשר‬
‫יכולות לעשותם מתאימים לשימושים כגון הנדסת רקמות ושחזורן‪ .‬תוצאות ראשונית אף מראות‪ ,‬שלנוכחות‬
‫הסיבים גם ערך כסמן טקסונומי‪-‬מורפולוגי לקבוצת מינים ממשפחת ה‪ ,Alcyoniidae -‬שהוצע שהיא מהווה‬
‫סוג קריפטי‪ .‬המחקר הנוכחי מהווה בסיס ידע‪ ,‬אשר יקדם תחומי מחקר מגוונים וידגיש את השילוב בין ממצאי‬
‫שדה ועבודת מחקר במעבדה למטרות מחקר בסיסי ויישומי כאחד‪.‬‬
‫עבודה זו נעשתה בהדרכת‬
‫פרופ' יהודה בניהו‬
‫תל‪-‬אביב‬
‫אוניברסיטת‬
‫הפקולטה למדעי החיים ע"ש ג'ורג' ס‪ .‬וייז‬
‫המחלקה לזואולוגיה‬
‫מאפיינים ביולוגיים‪ ,‬ביוכימיים ומכאניים של סיבי קולגן מהאלמוג‬
‫הרך בצקנית ‪Sarcophyton ehrenbergi‬‬
‫החיבור לשם קבלת תואר "דוקטור לפילוסופיה"‬
‫ע"י עדו סלע‬
‫הוגש לסנאט אוניברסיטת תל אביב‬
‫מרץ ‪2112‬‬