Structure, Physiology,

and Biochemistry of Collagens 2

Michael J. Mienaltowski and David E. Birk

Abstract
Tendons and ligaments are connective tissues that guide motion, share
loads, and transmit forces in a manner that is unique to each as well as the
anatomical site and biomechanical stresses to which they are subjected.
Collagens are the major molecular components of both tendons and
ligaments. The hierarchical structure of tendon and its functional properties
are determined by the collagens present, as well as their supramolecular
organization. There are 28 different types of collagen that assemble into
a variety of supramolecular structures. The assembly of specific supra-
molecular structures is dependent on the interaction with other matrix
molecules as well as the cellular elements. Multiple suprastructural assem-
blies are integrated to form the functional tendon/ligament. This chapter
begins with a discussion of collagen molecules. This is followed by a
definition of the supramolecular structures assembled by different
collagen types. The general principles involved in the assembly of
collagen-containing suprastructures are presented focusing on the
regulation of tendon collagen fibrillogenesis. Finally, site-specific differ-
ences are discussed. While generalizations can be made, differences exist
between different tendons as well as between tendons and ligaments.
Compositional differences will impact structure that in turn will deter-
mine functional differences. Elucidation of the unique physiology
and pathophysiology of different tendons and ligaments will require
an appreciation of the role compositional differences have on collagen
suprastructural assembly, tissue organization, and function.

M.J. Mienaltowski • D.E. Birk (*)

Departments of Molecular Pharmacology
& Physiology and Orthopaedics & Sports
Medicine, University of South Florida,
Morsani College of Medicine,
12901 Bruce B. Downs Blvd., MDC8,
Tampa, FL, 33612, USA
e-mail: [email protected]

J. Halper (ed.), Progress in Heritable Soft Connective Tissue Diseases, Advances in Experimental Medicine 5
and Biology 802, DOI 10.1007/978-94-007-7893-1_2, © Springer Science+Business Media Dordrecht 2014
6 M.J. Mienaltowski and D.E. Birk

Keywords
Hierarchical structure of tendon • Supramolecular structures of collagens
• Collagens I-XXVIII • Fibril-forming collagens • Procollagens • Triple
helix • Crosslinking • Fibril-associated collagens with interrupted triple
helices (FACIT) • Beaded filament-forming collagen • Network-forming
collagens

2.1 Introduction by a contiguous epitenon cover. Structure and

composition of such collagen-rich tissues allow
The composition and structure of tendons and for tendons and ligaments to be relatively compliant
ligaments play important roles in their functions. at low energy, low loading forces, yet to increase
Tendons attach muscles to bones, and ligaments in stiffness with increasing forces and loads.
act to connect bone to bone across a joint space. This chapter provides an overview of collagens
Both tendons and ligaments are fibrous connective with a focus on tendons and ligaments. The first
tissues that are composed of cells within an extra- section provides an overview of collagens focus-
cellular matrix rich in collagens, proteoglycans, ing on those collagens found within tendons
and water. However, the distinct function of and ligaments. This is followed by a discussion
each connective tissue is intricately linked to of collagen synthesis, fibril assembly, as well as
its specific composition and resulting structure. fibril growth, and maturation from the assembly
This also can be described for tendon type or of protofibrils to the organization of fibrils into
location (e.g., axial versus limb, flexor versus mature fibers. Finally, the tissue structure and
extensor) and for regions of tendons (e.g., mid- function of the collagen-rich extracellular matrix
substance versus enthesis). The composition of of connective tissues like tendons and ligaments
tendons and ligaments allows these connective are discussed in the context of differing roles for
tissues to help guide motion, to resist abnormal the collagens in musculoskeletal physiology.
displacement of bones and center the actions of
several muscles, and to share load and distribute
force. The general compositions of tendons and 2.2 Collagens
ligaments are similar. Both are composed of
water (50–60 % for tendons and 60–70 % for Collagens are proteins that are major components
ligaments), collagens (70–80 % dry weight for of the extracellular matrix of connective tissues.
tendons and more than 80 % for ligaments), Members of the collagen family are trimers. They
and proteoglycans, including small leucine-rich have at least one collagenous or COL domain as
proteoglycans [1]. The collagens present and well as non-collagenous or NC domains. The
the supramolecular structures assembled play number and structure of COL and NC domains
significant roles in the function of these musculo- are dependent upon the specific collagen type.
skeletal connective tissues. Tendons are hierar- The importance of these domains will be dis-
chical structures with structure and function cussed. Among the genomes of vertebrates and
being closely linked (Fig. 2.1). Tendons are higher invertebrates, there are 28 distinct collagen
composed primarily of collagen fibrils. Bundles glycoproteins that are encoded by at least 45
of fibrils are organized as fibers. Fibers are genes. These collagens have been given Roman
grouped together with tenocytes as fascicles, which numeral designations (I–XXVIII) in chronologi-
are surrounded by a cellular, loose connective cal order of discovery, and they are classified by
tissue, the endotenon. The outer edge of a bundle type based on domain structure and their supra-
of fascicles comprising the tendon is demarcated structural organization. For each collagen type,
2 Structure, Physiology, and Biochemistry of Collagens 7

Fig. 2.1 Hierarchical

structure of tendon.
Tendons are hierarchical
structures composed
primarily of collagen
fibrils. Triple helical
collagen molecules
assemble to form fibrils.
Fibrils bundle together to
form fibers. Within the
mature tendon, fibers are
grouped together with
tenocytes within fascicles
that are surrounded by a
cellular, loose connective
tissue, the endotenon. The
outer edge of a bundle
of fascicles is the epitenon;
it contiguously covers the
outside of the tendon

each genetically distinct alpha chain is designated and non-collagenous proteins. Collagens in each
by Arabic numerals. The alpha chains of one col- category have their own specialized function
lagen type are unique from the alpha chains of and contribute to higher order tissue structures.
another collagen type. Each distinct alpha chain is As collagens from various categories assemble
encoded by a different gene, and each distinct together with varying abundance and with co-
alpha chain has its own primary (domain) struc- polymerization with other non-collagenous
ture which contributes to the classification by col- macromolecules, they contribute to a tissue
lagen type. For example, the human α1(I) chain is suprastructure and thus to its function. Likewise,
encoded by the COL1A1 gene and the mouse with development and growth as well as repair
α1(II) chain by the Col2a1 gene. Collagens can be and remodeling, the relative abundance and
homotrimeric; that is, they are composed of three localization of different collagenous and non-
identical alpha chains, like [α1(II)]3 for collagen collagenous macromolecules are major determinants
II. However, collagens also can be heterotrimeric, of the structure and function of that tissue. This is
comprised of alpha chains encoded by different certainly true of tendons and ligaments.
genes of the same collagen type, like [α1(I)] In tendons and ligaments, greater than 90 % of
[α2(I)]2 for collagen I. Moreover, it is possible for the connective tissue is composed of collagen
a single collagen type to have multiple chain com- I [1]. While collagen I is the predominant collagen
positions, like [α1(V)] [α2(V)]2, [α1(V)] [α2(V)] in these tissues, other collagen types and non-
[α3(V)], or [α1(V)]3 for collagen V. collagenous macromolecules add diversity to the
Collagens may be grouped into classes based matrix and regulate just how fibril and fiber
upon their suprastructural organization (Table 2.1). assembly occurs. This ultimately leads to tissue-
Some collagens are fibril-forming; for example, specific structure and organization. As is the case
collagens I, II, and III. These collagens form with tendons by type, location, and even by
fibrils with a distinct 67 nm periodicity. There are zone within each tendon, collagen-containing
also Fibril-Associated Collagens with Interrupted suprastructures function differently based upon
Triple helices (FACIT) collagens that associate their complex macromolecular compositions
with collagen fibrils and interact with collagenous that include other collagen types as well as
8 M.J. Mienaltowski and D.E. Birk

Table 2.1 General classification of collagen types

Classification Collagen types Supramolecular structure
Fibril-forming collagen I, II, III Striated fibrils
V, XI Striated fibrils, retain
N-terminal regulatory domains
XXIV, XXVII Unknown
FACITa collagens IX, XII, XIV Associated with fibrils, other
interactions
FACIT-like collagens XVI, XIX, XXI, XXII Interfacial regions, basement
membrane zones
Network-forming collagens
Basement membrane IV Chicken wire network with
lateral association
Beaded filament-forming VI Beaded filaments, networks
Anchoring fibrils VII Laterally associated anti-parallel
dimers
Hexagonal networks VIII, X Hexagonal lattices
Transmembrane collagens XIII, XVII, XXIII, XXV Transmembrane and shed
Gliomedins, ectodysplasin soluble ecto-domains
Multiplexin collagens XV, XVIII Basement membranes, cleaved
(Endostatin–XV and -XVIII) C-terminal domains influence
angiogenesis
Other molecules with XXVI, XXVIII Collagenous domains in
collagenous domains Acetylcholinesterase, adiponectin, C1q, primarily non-collagenous
collectins, surfactant protein, others molecules
a
Fibril-associated collagen with interrupted triple helix

non-collagenous components. These additional type III collagen is found in greater abundance
macromolecules may be substantial or only during embryonic development; however, with
occur in minute quantities. However, typically maturation levels of collagen III decrease, though
the architecture and function of tendons and in the chicken it still persists in the endotenon
ligaments are determined by the composite struc- and tendon sheath [5]. Moreover, in mature ten-
ture of collagen suprastructures. In the following dons and ligaments of rabbits, it has been noted
section, the collagens contributing to the supra- that collagen III comprises 5 % and 10 %, respec-
structural organization of tendons and ligaments tively, of all collagen content [6]. Otherwise, col-
will be reviewed. lagen I is the predominant collagen of the tendon
and ligament mid-substance. It should be noted
that collagen II is generally found within the
2.3 Fibril-Forming Collagens fibrocartilaginous zone of the enthesis site for
both tendons and ligament [7, 8]. Collagens V
The fibril-forming collagen subfamily includes and XI are quantitatively minor collagens found
collagens I, II, III, V, XI, XXIV and XXVII. co-assembled with types I, II and III; they are
Collagens I, II, III, V, and XI have been found in found on the surfaceome (i.e., plasma membrane
tendons and in ligaments [1–3]. These collagens and pericellular matrix) of tendon fibroblasts [9].
have a long uninterrupted triple helical domain This fibril-forming subclass retains portions of
(ca. 300 nm). Fibril-forming collagen genes clus- the N-terminal propeptide and is involved in the
ter into three distinct subclasses[4] and this regulation of fibril assembly.[2] Collagens XXIV
carries over into functional subclasses. Collagens and XXVII make up the third subclass and have
I, II and III are the most abundant proteins in the differences relative to the other fibril-forming
vertebrate body and are the bulk components of collagen types including, shorter helical regions
all collagen fibrils. Within tendons and ligaments, that are interrupted. Their structural organization
2 Structure, Physiology, and Biochemistry of Collagens 9

and specific roles remain to be elucidated, and V and XI; as mentioned above, these regulatory
they have yet to be found within tendons and fibril-forming collagens are characterized by
ligaments. partial processing of N-propeptide domains. The
The fibril-forming collagens are synthesized N-propeptides have a flexible or hinge domain
and secreted as procollagens. Procollagens contain (NC2) between the triple helical domain (COL1)
a non-collagenous C-terminal propeptide and and a short triple helical domain (COL2). The
an N-terminal propeptide. The N-propeptide is N-terminal domain (NC3) is composed of a vari-
composed of several non-collagenous domains able domain and a proline/arginine-rich protein
and a short collagenous domain. The presence of (PARP) domain. Partial processing removes the
the propeptides prevents premature assembly PARP yet retains the hinge, COL2, and variable
of collagen molecules into fibrils. The initial domains [18–20]. The regulatory fibril-forming
assembly of collagen into fibrils is regulated by collagens co-assemble with the major fibril-forming
the processing of the propeptides which involves collagens in the heterotypic fibril; however, the
several enzymes. The C-propeptides are processed N-terminal domain of the regulatory fibril forming
by bone morphogenetic protein 1(BMP-1)/tol- collagens cannot be integrated into the staggered
loid proteinases or furin [10, 11]. The processing packing of the helical domains. The rigid COL2
of the N-propeptides involves certain members domain of the collagen V or XII molecule can
of the a-disintegrin-and-metalloproteinase-with- project toward the fibril surface in the gap region
thrombospondin-like-motifs family (ADAMTS of the assembled fibril (Fig. 2.2b). Recent findings
2, 3 and 14) as well as BMP-1 [12]. Propeptide have demonstrated that alteration in collagens V
processing enzymes have specificity for each dif- and/or XI affects tendon fibril assembly during
ferent collagen type.[10, 12] Propeptide process- tendon development. Changes include altered fibril
ing may be complete, thus leaving a collagen structure, decreased fibril number and abnormal
molecule with one large central triple helical fibril and fiber organization [21]. Thus, interactions
domain and terminal, short non-collagenous between the fibrillar collagens affect the organization
sequences termed the telopeptides, as is the case of collagen fibrils within collagen-rich tissues
for collagens I and II. However, with collagens like tendons and ligaments [22].
III, V, and XI, processing can be incomplete, with
the retention of a C-telopeptide and a partially
processed N-propeptide domain, which have 2.4 Fibril-Associated Collagens
been implicated in the regulation of fibrillogene- with Interrupted Triple
sis [13–17]. After propeptide processing, colla- Helices (FACIT)
gen molecules self-assemble to form striated
fibrils with a periodicity of 67 nm. Within each FACIT collagens closely interact with fibril-forming
fibril, collagen molecules arrange longitudinally collagens. These molecules affect the surface
in staggered arrays. Thus, a gap occurs between properties of fibrils as well as fibril packing.
the ends of neighboring molecules, and this gap- Collagens IX, XII, XIV and XX are FACIT col-
overlap structure is present in all collagen fibrils lagens. Type IX collagen is primarily found
with a 67 nm D-periodic banding pattern. This is interacting with collagen II. Collagen IX is also a
presented schematically in Fig. 2.2a. proteoglycan with covalently attached glycosami-
Collagen fibrils are heterotypic. That is, they noglycan side chains in cartilage [23]. This is also
are assembled from mixtures of two or more true with Type XII collagen [24]. Collagens XII
fibril-forming collagen types. In tendons and and XIV have been found throughout musculo-
ligaments, collagens I and III are the quantitatively skeletal connective tissues, including tendons and
major fibril-forming collagens with collagen II ligaments at various times during development;
present within fibrocartilaginous regions. Heterotypic [25, 26] collagen XIV has been found specifically
collagen fibrils of tendons and ligaments also at the bone-ligament interface in bovine entheses
contain quantitatively minor amounts of collagens [27] In general, FACIT collagens have short COL
10 M.J. Mienaltowski and D.E. Birk

Fig. 2.2 Fibril-forming collagens. (a) Fibril-forming characteristic alternating light and dark pattern represents the
collagens are synthesized as procollagens. Procollagens respective overlap and gap regions of the fibril. (b) Collagen
contain a central COL domain and flanking propeptide fibrils are heterotypic. That is, they are co-assembled
N-and C-terminal NC domains. Propeptides are pro- from quantitatively major fibril-forming collagens (e.g.,
cessed and the resulting collagen molecules assemble to I, II, or III) and regulatory fibril-forming collagens (V or
form striated fibrils. Each fibrillar collagen molecule is XI). Regulatory fibril-forming collagens have a partially
approximately 300 nm (4.4D) in length and 1.5 nm in processed N-terminal propeptide, retaining a non-collag-
diameter. Within the fibril, the collagen molecules are enous domain that must be in/on the gap region/fibril
staggered N to C in a pattern that gives rise to the surface. The heterotypic interaction is involved in nucle-
D-periodic repeat. At the bottom of the panel, a ation of fibril assembly (This figure has been adapted
D-periodic collagen fibril from tendon is presented. The from Birk and Bruckner [131])

domains interrupted by NC domains with an interface between tissues [33, 34], including to
N-terminal NC domain that projects into the some extent musculoskeletal tissues, particularly
interfibrillar space (Fig. 2.3). FACIT collagens those that are adjacent to or fed by vasculature.
have two C-terminal domains NC1 and COL1 For example, vasculature is found along the
that are believed to interact with the collagen I sheath, paratenon, and epitenon of tendons in a
fibrils. At the N-termini of FACIT collagens, the tendon-specific manner and basically along the
large globular NC domains protrude from the “epiligament,” or surrounding surface layer of
fibril surface [2]. FACIT collagens along the surface tissue for ligaments [1]. Throughout the body,
of fibrils have been shown affect fiber suprastruc- there are diverse networks of basement mem-
tures and tendon biomechanics [25, 26]. branes composed of many macromolecules that
The FACIT-like collagens have features in are anatomical site-dependent. Likewise, there
common with FACIT collagen, though they are are several subtypes of collagen IV which are
structurally and functionally unique. This FACIT- composed of different stoichiometries of 6 collagen
like group includes collagens XVI, XIX, XXI and IV-encoding genes COL4A1 through COL4A6
XXII, yet their roles in musculoskeletal connec- reviewed by Khoshnoodi et al. [35].
tive tissues have yet to be elucidated [28–32].

2.6 Beaded Filament-Forming

2.5 Basement Membrane Collagen
Collagen
Collagen VI is ubiquitous within connective tis-
Collagen IV is considered a basement membrane sue; it is found as an extensive filamentous net-
collagen. It is the collagenous component of an work with collagen fibrils, and is often enriched
integrated network of several matrix molecules in pericellular regions. Collagen VI can be
that form an extracellular matrix that defines the assembled into several different tissue forms,
2 Structure, Physiology, and Biochemistry of Collagens 11

Fig. 2.3 FACIT collagens associate with fibrils. (a) The N-terminal NC domain that projects into the inter-fibrillar
domain structures of FACIT collagens found in tendons and space. (b) The FACIT collagens all associate with the sur-
ligaments are illustrated. Note that all FACITs have alterna- face of collagen fibrils, including N-truncated isoforms due
tive spliced variants, and collagen XII can have glycosami- to alternative splicing in collagen XII. Collagen XII is capa-
noglycan chains attached covalently. The FACIT collagens ble of other non-fibril interactions (not shown) (This figure
have 2-3 COL domains and 3-4 NC domains with a large has been adapted from Birk and Bruckner [131])

including beaded microfibrils, broad banded humans[40–43] (See also Chap. 12 by Bushby).
structures and hexagonal networks [36–38]. In tendons, when collagen VI is removed via null
Collagen VI interacts with many extracellular Col6a1 mouse model, tenocyte expression
molecules including: collagens I, II, IV, XIV; changes due to a lack of cell-matrix interactions
microfibril-associated glycoprotein (MAGP-1); [42]. In tendons, the absence of collagen VI results
perlecan; decorin and biglycan; hyaluronan, hep- in increases in fibril density, significant reductions
arin and fibronectin, as well as integrins and in load and stiffness, increased matrix metallo-
the cell-surface proteoglycan NG2. Based on the proteinase activity, and overall dysfunctional
tissue-localization and large number of potential regulation of fibrillogenesis [42].
interactions, collagen VI has been proposed to Collagen VI is commonly formed as a hetero-
integrate different components of the extracellu- trimer composed of α1(VI), α2(VI) and α3(VI)
lar matrix, including cells [39]. Collagen VI also chains [39, 44]. Each monomer has a 105 nm tri-
may influence cell proliferation, apoptosis, ple helical domain with flanking N- and C-terminal
migration, and differentiation. Thus, collagen VI globular domains. The α3(VI) chain of the hetero-
is involved in the development of tissue-specific trimer can be processed extracellularly. In addi-
extracellular matrices, repair processes and in the tion, structural heterogeneity is introduced by
maintenance of tissue homeostasis. In musculo- alternative splicing of domains, primarily of the
skeletal tissue, collagen VI has proven to be α3(VI) N–terminal domain. Three additional α
essential; mutations have been shown to cause chains of type VI collagen have been described,
various forms of muscular dystrophy as well as α4(VI), α5(VI), α6(VI); these chains have high
proximal joint contractures involving tendons in homology with the α3(VI) chain and may form
12 M.J. Mienaltowski and D.E. Birk

Fig. 2.4 Assembly of collagen VI suprastructures. of 3 different collagen VI suprastructures: beaded fila-
Collagen VI monomers have a C-terminal NC domain, ments, broad banded fibrils and hexagonal lattices.
a central triple helical domain, and an N-terminal NC These suprastructures form via end-to-end interactions
domain. The monomers assemble N-C to form dimers. of tetramers and varying degrees of lateral association
Tetramers assemble from two dimers aligned in-register. (This figure has been adapted from Birk and Bruckner
The tetramers are secreted and form the building blocks [131])

additional isoforms [45, 46]. The supramolecular mers, also intracellularly. The tetramers are
assembly of collagen VI begins intracellularly secreted and associate end-to-end to form beaded
(Fig. 2.4). Two collagen VI monomers assemble filaments extracellularly. The newly formed thin,
in a lateral, anti-parallel fashion to form a dimer; beaded filaments (3–10 nm) have a periodicity of
the monomers are staggered by 30 nm with the approximately 100 nm; they laterally associate to
C-terminal domains interacting with the helical form beaded microfibrils [36], and they are found
domains. The resulting overlap generates a central in hexagonal lattices [49]. The broad banded
75 nm helical domain flanked by a non-over- fibrils represent continued lateral growth of
lapped region with the N– and C-globular beaded microfibrils and/or lateral association of
domains, each about 30 nm. The C-terminal preformed beaded microfibrils. In contrast, hex-
domain-helical domain interactions are stabilized agonal lattices are formed via end-to-end interac-
by disulfide bonds near the ends of each over- tions of tetramers in a non-linear fashion [49].
lapped region [47]. The overlapped helices form Like fibrillar collagen structures, collagen
into a supercoil of the two monomers in the central VI-rich supramolecular aggregates are composite
region [48]. Two dimers then align to form, tetra- structures with other integrated molecules that
2 Structure, Physiology, and Biochemistry of Collagens 13

modulate the functional properties of the ultimate

suprastructure. For example, biglycan interactions 2.9 Procollagen Synthesis,
with the collagen VI tetramer induced formation Collagen Fibril Assembly,
of hexagonal lattices, instead of beaded microfi- Growth and Maturation
brils; though, decorin, which binds to the same
site, was less effective in inducing hexagonal Collagen synthesis, assembly, and maturation
lattice formation [49]. Thus, analogous to fibril require a sequence of well controlled intracellular
formation, the interaction of small leucine-rich and extracellular events. Collagen genes are
proteoglycans with collagen VI can influence the transcribed from DNA into mRNA within the
structure of the tissue aggregate and therefore its nucleus. Transcripts are transported out of the
function. The interaction of collagens with such nucleus and translated into procollagen mono-
molecules in a multitude of ways allows for the mers, which are post-translationally modified in
assembly of different suprastructures in adjacent the rough endoplasmic reticulum prior to assembly
regions or tissues with different functions, even into procollagen triple helices. The extent of
sometimes as simply as with the addition or these modifications is affected by the rate of
removal of non-collagenous molecules. triple helix formation, which is in turn affected
by the primary structure of the alpha chain pro-
peptides. For example, point mutations in the
2.7 Network-Forming Collagens Gly-X-Y sequence may result in altered molecular
properties for that chain as well as dysfunctional
Collagens VIII and X are closely related short regulation of chain selection, helix formation, or
chain collagens, with comparable gene and protein post-translational modification. These molecules
structures [39, 50]. While collagen VIII is not are then secreted as procollagens, which prevents
typically found in musculoskeletal tissues, collagen premature molecular assembly into suprastruc-
X has a very restricted distribution, found only in tures. Procollagens are then processed extracel-
hypertrophic cartilage. This collagen is a homotri- lularly, in most cases, and require collagen
mer composed of α1(X) chains and the supramo- type-specific metalloproteinases. Processing may,
lecular form is a hexagonal lattice [51]. Collagen however, begin during the transport of newly
X was recently reviewed [52]. synthesized procollagens to the cell surface [55,
56]. Processed collagen triples helices are then
cross-linked. The relationship between protein
2.8 Transmembrane Collagens structure and triple helix assembly and collagen
fibril formation will be discussed below, as well
Transmembrane collagens include collagens XIII, as the growth and maturation of fibrils in tendons
XVII, XXIII, and XXV. They are all homotrimers and ligaments.
and contain an N-terminal cytoplasmic domain
and a large C-terminal domain containing multiple
COL domains with NC interruptions providing 2.10 Triple Helix Assembly
flexibility. Transmembrane collagens are so classi- and the Impact of Primary
fied because they have a hydrophobic membrane Structure on Secondary,
spanning domain. Between this domain and an Tertiary, and Quaternary
adjacent extracellular linker domain is the first Structures
COL domain involved in trimerization which is
also subject to proteolytic cleavage generating a After collagen pre-pro-peptides have been trans-
shed extracellular domain. Of the transmembrane lated, they are directed into the lumen of the rough
collagens, collagen XIII is found in musculoskel- endoplasmic reticulum (RER). Modifications in
etal tissues, particularly in myotendinous and the pre-pro-peptide include cleavage of the
neuromuscular junctions [53, 54]. N-terminal signal for transport to the RER so that
14 M.J. Mienaltowski and D.E. Birk

it becomes a propeptide, or a pro-alpha chain As post-translational modification and triple

[57]. Pro-alpha chains undergo hydroxylation of helix assembly occur concomitantly, once initi-
prolyl and lysyl residues followed by glycosyl- ated, trimerization must be controlled to allow
ation of hydroxylysyl residues. Three pro-alpha for alpha chain post-translational events like
chains will trimerize; this involves the selection hydroxylation and glycosylation to occur. After
and alignment of appropriate alpha chains with or as collagen alpha chains are translated, amino
subsequent assembly into specific trimeric acids within the triple helical domains of each
collagen molecules [58]. Trimerization is initiated chain can be modified. Co-translational modifi-
through interactions among the non-collagenous cation of these domains includes hydroxylation
trimerization domains of alpha chains at their and glycosylation. Collagen polypeptides contain
C-termini in the RER [57]. two unique amino acids, hydroxyproline and
The primary amino acid sequence of collagen hydroxylysine, which are important downstream
alpha chains affects the protein structure at all for triple helix stability and glycosylation,
other levels. The primary structure of the collagen respectively. As the propeptides are synthesized,
alpha chain features a COL domain that will these unique amino acids are introduced by
coil into a left-handed helix lacking intra-chain enzymatic hydroxylation of almost all prolyl- and
hydrogen bonds. Three alpha chains will super- some of the lysyl residues in the Y-positions.
coil to form a triple helix. This parallel, right- Hydroxyproline residues are important in triple
handed superhelix is stabilized by inter-chain helix stability. The 4-trans hydroxyl group assists
hydrogen bonds that are almost perpendicular in directing the free hydroxyproline, similar to
to the triple helical axis. Because of the imino that of Y-hydroxyproline in collagen triple
acid content, collagen polypeptides assume an helices. The forced integration of proline into
elongated polyproline II-like helix with all Y-positions absorbs more ring deformation when
peptide bonds in the trans configuration. The compared with physiological hydroxylated
pitch of the polyproline II helix in collagenous collagen [63, 64]. Collagens are further modified
polypeptides corresponds to three amino acids, post-translationally after secretion and during
almost exactly. Steric constraints require that aggregate assembly.
only glycine, the smallest amino acid, can occupy Fibril structure also is affected by post-
the positions at the center of the triple helix. translational glycosylation of collagens. Covalent
Thus, the triple helical domains have a repeating modifications occurring after polypeptide syn-
(Gly-X-Y)n structure with the X and Y positions thesis are important in collagens and have an
being any of the 21 proteinogenic amino acids impact on fibril assembly [65–67]. The circum-
of the universal genetic code. However, the X ference of collagen triple helical domains is
and Y positions are typically occupied by proline affected by the extent of hydroxylation of lysyl
and hydroxyproline, which are necessary for residues and subsequent galactosylation and
helix formation and stability, respectively. glucosyl-galactosylation of hydroxylysyl residues.
Replacement of the glycine residues with other Intermolecular center-to-center distances correlate
amino acids interrupts the triple helix motif and with the extent of glycosylation, particularly if
causes the rod-like structures to have rigid post-translational modifications affect those
kinks or flexible hinges [59, 60]. This occurs in regions of polypeptides situated in overlap regions
many collagen types with primary structures of the fibril. The extent of glycosylation of
containing more than one helical domain thus hydroxylysine modifications can be manipulated
providing flexibility, e.g., in collagen V. However, by features such as increased enzyme activity lev-
the substitution of glycine residues also may be els[68] and disease-causing mutations [62]. Rates
the result of missense mutations in collagen of procollagen triple helix formation can be
genes and manifest as the underlying etiology substantially reduced in the rough endoplasmic
for mild or severe, systemic connective tissue reticulum by collagen mutations. Over-modification
diseases [61, 62]. leads to compromised fibrillar organization. Yet
2 Structure, Physiology, and Biochemistry of Collagens 15

different extents of glycosylation can serve as a 3-hydroxylase and Crtap, respectively, cause
physiological mode of regulation in normal severe recessive osteogenesis imperfecta [74].
native tissue, particularly when levels of post- Additional modifications occur post-translati
translational modification are tissue-specific and onally after secretion of procollagen and during
thus contribute to the structural properties of supramolecular assembly. Extracellular lysyl
tissues. Intrafibrillar water also has been shown oxidases can convert the amino groups on some
to affect the molecular organization of collagen of the hydroxylysine- and lysine residues in the
fibrils. As variable amounts of water are incorpo- collagen polypeptide chain to aldehydes that
rated within the fibrils, intermolecular distances form aldols or β-ketoamines [57]. This happens
between lateral or longitudinal neighbors have when the aldehydes of hydoxylysine or lysine
been shown to differ [69, 70]. When collagen residues react with aldehydes or amino groups on
fibrils are dried, their D-periodicity shortens lysines, respectively, in other chains to generate
intermolecular lateral distances reduce. intra- and inter-molecular covalent crosslinks
Collagenous domains of alpha chains are [75–77]. In some cases, cross-linking of collagen
distinctly rich in cis-peptide bonds due to their molecules at early stages of aggregation can
high content of imino acids that favor cis-peptide modulate the suprastructural outcome of fibrillo-
bond formation. Kinetically, a great deal of genesis or the formation of networks.
energy is required to cause cis-peptide bonds to
comply with triple helix formation. Isomerization
of each cis-peptide bond encountered is necessary 2.11 Collagen Fibril Assembly
during collagen triple helix formation which is a
zipper-like process. Helix assembly thus involves The assembly and deposition of collagen fibrils
a slow folding process when compared to other with tissue-specific structures and organizations
proteins [71, 72]. At the start of triple helix involves a sequence of events that occur in both
formation, a variable number of cis bonds are intracellular and extracellular compartments.
distributed throughout the still unfolded procol- Collagen molecules are synthesized, hydroxyl-
lagen polypeptides. Hence, the folding times ated, glycosylated, assembled from three poly-
required for full-length triple helix formation are peptides, and folded in the rough endoplasmic
heterogeneous depending upon kinetic obsta- reticulum. Then packaging of the trimers occurs
cles. In fibroblasts, the cis to trans isomerization in the Golgi, and transport is via specialized and
of Gly-Pro-, but not X-Hyp peptide bonds, is elongated intracellular compartments with secre-
catalyzed by cyclophilin B, which acts as pepti- tion at the cell surface. Collagen fibrils are
dyl prolyl cis/trans-isomerase. Cyclophilin B composites of different matrix molecules, and
can be inhibited by the immuno-suppressor control of heteropolymeric mixing and trimer
cyclosporin A [73]. Moreover, cyclophilin B type stoichiometry begins within the intracellular
along with prolyl-3-hydroxylase and cartilage- compartments. Moreover, secretion of different
associated protein (Crtap) form a ternary com- matrix molecules and modifying enzymes occurs
plex with high chaperone activity in the with specific spatial, temporal, and circumstan-
endoplasmic reticulum. Prolyl-3-hydroxylase tial patterns. Therefore, the character of the
introduces a single 3-Hyp-residue at the assembled fibrillar matrix depends upon not only
C-terminal end of the triple helical domain of the collagen types synthesized, but on the regu-
nascent fibrillar procollagens. This ternary com- lated interactions with procollagen processing
plex localizes cyclophilin B-activity to the initiation enzymes, fibril-associated molecules (e.g., pro-
sites for procollagen folding. This allows for teoglycans and FACITs), and adhesive glycopro-
efficient catalytic isomerization of peptidyl-prolyl teins. The spatial and temporal regulation of
cis bonds. Such a role is supported by data mixing during packaging and transport or at the
from humans where null mutations in LEPRE1 sites of secretion provides a mechanism whereby
and CRTAP, the genes encoding human prolyl- limited numbers of matrix molecules can be
16 M.J. Mienaltowski and D.E. Birk

assembled to produce the diversity of structure extracellular matrix as small bundles of fibrils or
and function observed across tissues. immature fibers. There they are stabilized via
Extracellularly in the developing tendon, fibril interactions with macromolecules such as FACITs
assembly begins in deep recesses or channels and small leucine-rich proteoglycans (Fig. 2.6).
defined by the fibroblast surface [55, 78–80]. In Tendon maturation continues with linear fibril
these micro-domains, protofibrils are assembled growth involving end overlap of the protofibrils,
[78, 81]. These immature fibrils have small and followed by lateral growth in tendons and liga-
uniform diameters as well as short lengths ments. Lateral fibril growth features the associa-
compared to mature fibrils. These extracellular tion and fusion of fibrils laterally to generate
channels have been shown to form at the time of larger diameter fibrils. This process involves
secretion as specialized post-Golgi secretory molecular rearrangement of fibrils so that a cylin-
compartments, fuse with the fibroblast membrane drical fibril structure is generated. To accomplish
and are maintained due to slow membrane recy- this, some or all of the collagen stabilizing com-
cling associated with the presence of the assem- ponents stabilizing are lost or replaced.
bled protofibril [78, 81]. Other data also suggest Throughout this process, the number of intra- and
that intracellular processing of procollagen may intermolecular crosslinking plays an important
occur within elongated Golgi-to-plasma mem- role in regulation of structure turnover and stabil-
brane compartments (GPCs); this is followed by ity. Increases in stability generated by crosslink-
the extrusion of protofibrils through a cellular ing improve mechanical stability that varies by
protrusion, where GPC fuse to fibroblast plasma location within tendons and ligament and ana-
membranes [55, 79]. Either way, protofibrils are tomically throughout the body. The specific roles
ultimately present extracellularly. Once the of collagen accretion in fibril assembly/repair
protofibrils are deposited into the extracellular during regeneration remain to be elucidated.
matrix, more compartmentalization occurs. That Moreover, contributions in tissue homeostasis
is, fibrils form small fibers and then larger and with normal turnover and repair are likely.
structures characteristic of the specific tissue
(e.g., large fibers in tendon). This compartmen-
talized hierarchy within the extracellular allows 2.13 Regulation of Collagen Fibril
the fibroblast to exert control over the extracel- Assembly and Growth
lular steps of matrix assembly (Fig. 2.5).
Regulation of collagen fibrillogenesis is tissue-
specific. Within each tissue, interactions occur
2.12 Assembly and Growth amongst many different classes of molecules,
of Mature Tendon such as processing enzymes, heterotypic
Collagen Fibrils fibril-forming collagens, FACITs, and small
leucine-rich proteoglycans (SLRPs) as well as
In mature tendon, collagen fibrils are functionally other glycoproteins, such as, fibronectin, tena-
continuous. That is, fibrils are long, though sin X, etc. Many regulatory interactions occur
lengths unmeasured, and have diameters ranging and one way to impose some order on the discus-
between 20 and 500 nm depending on the tissue sion is to consider three classes of interactions;
and developmental stage [82–84]. However, molecules that act as organizers, nucleators, and
during tendon development collagen fibrils are regulators [87].
assembled near the fibroblast surface as uniform For example, significant increases in collagen
and relatively short D periodic protofibrils with fibril diameters occur during growth and develop-
diameters in the range of 20–40 nm, lengths of ment of tendon, and mechanical properties of the
4–12 μm, and tapered ends [81, 82, 85, 86]. tendon are dependent on increases in fibril diam-
The newly assembled protofibrils are deposited eter seen.[88] Tendon fibroblasts express collagen
and incorporated into the developing tendon I as the major fibril-forming collagen with minor
2 Structure, Physiology, and Biochemistry of Collagens 17

Fig. 2.5 Extracellular compartmentalization of fibril and deposited into the developing matrix in fiber-forming spaces
fiber assembly by tenocytes. In the developing chicken ten- where fibers coalesce to form fibers. Fibers continue to
don, extracellular compartmentalization of the different levels become larger as a result of the aggregation of adjacent fibers
of matrix assembly is seen in both panels (a) and (b). These in a third domain. As cytoplasmic processes that define fiber-
panels were generated from cross-sections of 14-day chicken forming compartments retract (curved arrow), fibers (fibril
embryo tendon cut perpendicular to the tendon axis A series bundles) are allowed to coalesce into larger aggregates
of micro-domains are evident. Protofibrils are assembled in characteristic of mature tissue. Bar, 1 micrometer (Modified
fibril-forming channels (arrowheads). Protofibrils are from Birk and Linsenmayer 1994)

quantities of collagen V. Thus, tendon fibrils are lethal due to a lack of fibril formation in the
heterotypic, containing collagens I and V. mesenchyme; though collagen I is synthesized
Collagens V and XI have been shown to and secreted, protofibrils are not properly assem-
nucleate collagen fibril formation in self-assembly bled [91]. Similarly, in two separate mouse models
assays [89–92]. The alpha-chains of collagens V where collagen XI is absent (cho/cho, ablated
and XI are highly homologous; thus, these colla- Col11a1 alleles; Col2a1-null mice, from which
gens represent different isoforms of a single the α3(XI) chain is derived), animals develop
collagen type. A mouse model with a targeted chondrodysplasia (cho) where cartilage is devoid
deletion within the Col5a1 gene is embryonic of fibrils.[93–96] These data demonstrate a key
18 M.J. Mienaltowski and D.E. Birk

Fig. 2.6 Model of the regulation of fibril assembly. syndecans) at the cell surface. (b) Protofibrils are deposited
Model of the regulation of fibril assembly. Fibril assembly into the matrix and are stabilized by interactions with regula-
involves a sequence of events. (a) First, nucleators (e.g., col- tors – other matrix components such as SLRPs and
lagen V and XI) initiate fibril assembly at the fibroblast cell FACITs. (c) Changes in fibril stabilization resulting from
surface. Then immature, small diameter, short protofibrils processing, turnover and/or displacement regulate linear and
are assembled. The nucleation process is cell directed lateral growth to mature fibrils in a tissue-specific manner
involving interactions with organizers (e.g., integrins and (This figure has been adapted from Birk and Bruckner [131])

role for collagen V/XI in nucleation of assembly fibroblast to define the site of fibril formation.
of short, small diameter protofibrils. That is, the number of originating fibrils is
Under physiological conditions in vitro, col- controlled by the number of nucleation sites
lagens I and II can self-assemble after long lag provided by the fibroblast. This feature is tissue-
phases. The nucleation of fibril formation by specific. For example, in the cornea where smaller
collagen V or XI provides a mechanism for the fibril diameters are paramount to transparency,
2 Structure, Physiology, and Biochemistry of Collagens 19

collagen V is 10–20 % of the fibril-forming col- other extracellular matrix molecules. Fibronectin
lagen content, and the many nucleation sites con- molecules assemble into fibrils via integrin inter-
tribute to many smaller diameter fibrils [97, 98]. actions [87, 100, 101]. The fibronectin fibril
However, in tendons where mechanical strength network contains multiple binding sites for
depends upon larger diameter fibrils, collagen V collagen fibril assembly. When binding sites are
makes up 1–5 % of the fibril-forming collagen blocked in fibronectin networks, collagen fibril
content. One classic example of the collagen I/V assembly is inhibited [102]. Moreover, modifica-
interaction and its impact on structure is best seen tions to fibronectin-integrin interaction affect
in the classic form of Ehlers-Danlos Syndrome, a collagen fibril assembly, suggesting both that the
generalized connective tissue disorder where the cytoskeleton is involved in some way with collagen
majority of patients are heterozygous for muta- fibril assembly and that other direct interactions
tions in collagen V [99]. This disorder results in of integrins with other surface molecules are
approximately 50 % of the normal collagen V. essential to fibril assembly.
Affected patients have a dermal phenotype with Once the protofibrils are assembled and
large, structurally aberrant fibrils. Similarly, in deposited into the extracellular matrix, further
the heterozygous (Col5a1+/−) mouse model, assembly to the mature fibrils involves linear
there was a reduction of 50 % in collagen V fewer and lateral growth of the preformed intermedi-
fibrils assembled, indicating fewer nucleation ates. In tendons and ligaments, numerous mole-
events than the normal mice [91], a similar situa- cules are involved in the regulation of these
tion occurs in the tendon [21]. The reduction in steps. Two classes of regulatory molecules are
nucleation sites makes collagen V a rate limiting the small leucine-rich proteoglycans (SLRPs)
molecule in this instance. The regulation of these and the FACIT collagens. Both classes are fibril-
collagen I/V interactions is coordinated by the associated and molecules within each class have
domain structure at the sites of assembly and by their own tissue-specific, temporal, and spatial
other molecules that organize and sequester these expression patterns. Differences in expression
interactions at the cell surface [9]. patterns contribute to the differences in structure
The nucleation of protofibrils involves direc- and function amongst tissues, including tendons
tion from cellular structures such as organelles, and ligaments.
cytoskeletal components, cytoplasmic membrane Small leucine rich proteoglycans (SLRPs) are
domains, as well as organizing molecules. important regulators of linear and lateral fibril
However, equally important are cell-defined growth [103]. Two classes of SLRPs are
extracellular domains. Without such direction, expressed throughout tendon growth and matura-
fibril assembly is inefficient. For example, in tion. They are divided into class I (decorin and
monolayer cultures, in the absence of extracel- biglycan) and class II (fibromodulin and lumican).
lular matrix, procollagens are secreted freely When the genes for these molecules are specifi-
into the media. Thus, organizing molecules cally targeted via single or compound SLRP
provide a resource for tissue-specific coupling of deficient mice, their importance in regulation of
fibril assembly to the cell surface. Consequently, linear and lateral fibril growth in tendons is
cell-directed positioning of the deposited matrix demonstrated [103–109]. In tendons, decorin and
is possible as assembled protofibrils undock fibromodulin are dominant in this regulation, and
from the cell surface, are extruded from the cell, they can be modulated by biglycan and lumican,
and are incorporated into the extracellular matrix respectively [88, 110]. A lack of decorin, bigly-
so that nucleation sites might be re-primed for can, or fibromodulin leads to disruptions in fibril
the next round of nucleation. growth, resulting in larger diameter fibers,
Cell-directed collagen fibril assembly involves structural abnormalities, and biomechanical
organizing molecules such as integrins and fibro- alterations in the tendon [88, 104, 111–114].
nectin. Fibronectin mediates the cell’s interaction Moreover, synergistic (additive) effects are seen
with assembling collagen fibrils as well as with between classes when compound biglycan and
20 M.J. Mienaltowski and D.E. Birk

fibromodulin deficiencies occur that affect fibril tendon is crucial for transmitting the force that is
diameters, alter tendon biomechanics, and even generated from muscle to bone. In contrast, the
promote ectopic ossification within the tendon ligament has a more passive role of attaching
[104, 111, 115]. Unlike fibril-forming collagens, bone to bone, guiding the joints motion, and
SLRPs regularly turnover and more easily allow preventing abnormal displacement of bones; to a
for changes in expression to affect fibrillogenesis minor extent, ligaments must also withstand load
and tendon structure throughout development, to resist joint instability. Collagens make up
maturation, and injury. A more detailed descrip- 60–70 % and 70–80 % of the dry weight for liga-
tion of activities mediated by SLRPs can be ments and tendons, respectively [1, 116]. The
found in Chap. 4. predominant fibril-forming collagen of tendons
The regulation of linear and lateral fibril and ligaments is collagen I; other collagens
growth is also affected by FACIT collagens. As within tendons and ligaments include III, V, VI,
described earlier in the chapter, FACIT collagens XII, and XIV. Besides collagens, tendons and
are fibril-associated molecules with large non- ligaments contain many of the macromolecules
collagenous domains. Like SLRPs, FACITs also mentioned throughout this chapter that regulate
demonstrate differing tissue-specific and temporal fibril assembly as well as water (60–70 % in
expression patterns. Collagen IX is involved in ligaments and 50–60 % in tendons) [116]. Thus,
regulation of fibril growth in cartilage [23]. while the content of these tissues is quite similar,
Collagen XIV has recently been implicated in the there are distinctions in proportional composi-
regulation of tendon fibril growth [25]. Targeted tions of water and macromolecules. Likewise,
deletion of collagen XIV in mouse models dem- there are several similarities in the morphologies
onstrated a premature entrance into the fibril of the two tissues. Both tendons and ligaments
growth stage in tendons, resulting in larger diam- have many parallel fibers that run along the axis
eter fibrils in early developmental stages. This of tension and these fibers are composed of
indicates that in some tissues like tendons mainly fibrils containing collagen I. There is also
FACITs may serve as ‘gate keepers’ regulating a “crimping” pattern in the collagen fibers of both
the transition from protofibril assembly to fibril tissues. However, there are several morphologi-
growth during development. In the case of collagen cal distinctions. For example, within ligaments
XIV, it temporarily stabilizes protofibrils to there are many regions where collagen fibers
prevent the initiation of lateral fibril growth. The intertwine with adjacent fibers so that subsets of
large non-collagenous domain and its inter-fibrillar fibers are running obliquely at a 20–30° angle
location have been long suspected to play an from the other fibers running along one axis
additional role in fibril packing. Control of fibril [117]. These differences in composition and
packing also would influence lateral associations morphology are enough to contribute to the
necessary for growth. function of the tissues.
Composition of both tendons and ligaments
contributes to the nonlinear anisotropic mechanical
2.14 Effects of Composition behavior that is exhibited in both tissues. An
and Structure on Function example load-elongation curve is depicted in
for Tendons and Ligaments Fig. 2.7. When loading conditions are low, tendons
and ligaments are relatively compliant. In the
Tendons and ligaments are dense connective “toe” region of the curve, initial load is affecting
tissues composed of similar proteins and other the “crimped” collagen fibers and any viscoelas-
macromolecules. Both tissues have a large extra- tic properties provided by molecules interacting
cellular matrix to cell ratio. However, slight with the collagens. The “toe” regions of the ten-
differences in content and morphology allow for don is smaller because most collagen fibers are
each tissue type to function in a distinct manner. oriented parallel to the directions of strain and
The composition of the extracellular matrix in thus less realignment is necessary. With increasing
2 Structure, Physiology, and Biochemistry of Collagens 21

Fig. 2.7 Load-elongation curve for tendons and as the tendon or ligament becomes increasingly stiffer.
ligaments. There are three distinct regions within the In this region, load/elongation will ultimately follow a
curve which define the response to tensile loading. In linear slope as slippage occurs within and then between
the “Toe Region,” initial load is affecting the “crimped” collagen fibrils. In the “Failure Region,” load continues
collagen fibers and any viscoelastic properties provided to increase until the point of tearing as adjacent fibril
by molecules interacting with the collagens. In the molecules slip away to tensile failure, or complete
“Linear Region,” increasing tensile loads cause lengthening rupture

tensile loads, these tissues – tendons more so is generally greater than that of a ligament
than ligaments – become increasingly stiffer. At (meniscofemoral ligament, 355 ± 234 MPa;
some point, load/length, or stiffness, will follow a anterolateral bundle of the posterior cruciate
linear slope as slippage occurs within collagen ligament, 294 ± 115 MPa; posterior bundle of the
fibrils, next between fibrils, and then until the posterior cruciate ligament, 150 ± 69 MPa) [118,
point of tearing as adjacent fibril molecules slip 119]. Moreover the stiffness for tendons (Achilles
away with tensile failure (termed, ultimate load tendon 430 N/mm) is generally greater than for
or point of ultimate tensile stress). As the load ligaments (lateral/medial collateral ligaments,
proceeds from initial strain to the point of failure, 20 N/mm; anterior cruciate ligament, ACL
the area under the curve is considered the total 182 N/mm) [120–122]. Greater values for elastic
energy absorbed. modulus and stiffness are indicative of stiffer,
Though both tendons and ligaments are con- less flexible connective tissue that is capable of
sidered to exhibit a nonlinear anisotropic absorbing and transmitting more energy. In addi-
mechanical behavior, differences in their load- tion, the tensile strength of tendons (50–150 N/m2)
elongation curves do exist. As already mentioned, is greater than that of ligaments (26–39 N/m2)
the morphology of the ligament allows for the [123–125]. The compositional differences and
“toe” of the curve from the initial loading to be distinctions in stiffness and tensile strength
longer because the crimping pattern of the between tendons and ligaments all contribute to
ligament is more greatly affected by fibrils not the understanding of how these two connective
oriented exactly parallel – sometimes even per- tissues function.
pendicular to the direction of the load. The stiffness Tendons and ligaments have different roles.
of tendons and ligaments are also distinct. In Tendons center the actions of several muscles
humans, the elastic modulus of a tendon into one axis of stress or strain. They distribute
(Achilles tendon, 375 ± 102 MPa; biceps tendon, contractile force of muscles to bones, and they
421 ± 212 MPa; patellar tendon, 660 ± 266 MPa) provide the muscle with distance from the insertion
22 M.J. Mienaltowski and D.E. Birk

that might mechanically beneficial. Tendons location [1]. Tendons like that of the flexor
store elastic energy during locomotion, and digitorus profundus are round and contain the
prevent muscle injury with viscoelasticity. The typical parallel bundles of collagen fibers.
relative stiffness and tensile strength of tendons is However, tendons such as those in the rotator cuff
essential for maintaining force transmission. are flat, layered, and multi-directional; each
Ligaments, on the other hand, guide joint motion tendon contains parallel collagen fibers as well as
by attaching adjacent bones involved. This stabi- fibers that interdigitate obliquely with fibers from
lizes the joint and controls the range of motion other tendons within the rotator cuff [126].
when load is applied. Their flexibility, relative to Rotator cuff tendons also contain more proteo-
tendons, allows for range of motion within the glycans throughout than the typical round ten-
joint. Tendons are susceptible to injury from dons; additional proteoglycans are believed to be
overuse, wear and tear, and abrupt tears or avul- aggrecan and SLRP biglycan [127]. A study of
sions when great forces are applied. Ligaments, ovine tendons and ligaments demonstrated in an
though flexible, have less tensile strength and are extracellular matrix analysis that each tendon
prone to shear force injuries. The functions of (long digital extensor tendon, superficial digital
tendons and ligaments are made manifest by flexor tendon, patellar tendon) and ligament (lat-
thorough consideration of their composition, eral collateral ligament, medial collateral liga-
morphology, and physiology. ment, posterior cruciate ligament, anterior
While tendons and ligaments possess charac- cruciate ligament) had its own unique range for
teristics that might distinguish one connective matrix compositions when examining water, gly-
tissue from another, each of these tissue types cosaminoglycan, and collagen content [116].
also differs by anatomical location. For example, Moreover, each tendon and ligament demon-
features of an Achilles tendon are not identical to strates its own collagen organizational and
those of a flexor digitorus profundus tendon or a mechanical features [116]. Gross anatomical dif-
patellar tendon. Likewise, characteristics of an ferences in tendon and ligament size and shape
anterior cruciate ligament are not identical to occur as these connective tissues: traverse areas
those of a medial collateral ligament. In addition, with limited space (e.g., within the wrist), cen-
within each tendon and ligament, there are zones tralize the force of several muscles (e.g., the
where composition changes. For example, liga- Achilles tendon), or manage multi-directional
ments can be divided into ligament mid-substance, forces and movements by intertwining collagen
fibrocartilage, mineralized fibrocartilage, and fibers with fibers of nearby connective tissues
bone. Similarly, tendons have musculotendinous associated with tension in another axis (e.g., cru-
junctions, mid-substance, fibrocartilage, and ciate ligaments and rotator cuff tendons). The
mineralized fibrocartilage to the enthesis. In the precise composition and structural arrangement
following sections, differences in collagen structure of each tendon and ligament provides specific
and physiology will be described by anatomic mechanical properties to allow that connective
location and by zone. tissue to function. Thus, to some extent, each ten-
don and ligament has its own unique features.

2.15 Effect of Anatomical

Location on Tendons 2.16 Roles of Collagens
and Ligaments in Transition from
Midsubstance to Enthesis
While most tendons and ligaments are generally in Tendons and Ligaments
composed of the same content described through-
out the chapter, there are slight differences in Much of what has been described in this chap-
gross structure and content that might better ter to this point has covered the tendon and liga-
allow the tendon to function at its anatomical ment mid-substance. The transition of tendons
2 Structure, Physiology, and Biochemistry of Collagens 23

Fig. 2.8 Transition zones. This illustration demonstrates cartilage and the next zone, calcified fibrocartilage.
the transition from fibrous tissue to bone at a ligament Calcified fibrocartilage interdigitates with the underlying
insertion. The tissue transitions from ligament to subchondral bone to complete the insertion. Structures of
fibrocartilage. Increases in the level of calcification are note within the illustration include: ligament fibroblasts
noted in the compositional gradient closer to insertion; (LF), fibrocartilage chondrocytes (Ch), osteoblasts (Ob),
this compositional change is demarcated by a tidemark (T) Sharpey’s fibers (SF), and blood vessels (BV) (This figure
which traces the interface between non-calcified fibro- has been adapted from Place et al. [132])

and ligaments toward entheses are associated as aggrecan and decorin [ 129 ]. The composi-
with changes in their composition and organi- tion of the second zone departs from that of the
zation. There is a transition from tendinous and tendon or ligament-midsubstance. The third zone
ligamentous material to bone (Fig. 2.8). These contains mineralized or calcified fibrocartilage. It
transition sites are not simple discreet units; is primarily composed of collagen II with signifi-
instead, there is more of a gradient of molecular cant amounts of collagen X and aggrecan [130].
differences from mid-substance to bone. That The fourth zone is characterized as bone; it is
said, one can identify general zones: mid-substance, predominantly composed of collagen I as well as
fibrocartilage, calcified fibrocartilage, and bone components typically found within bone. The
[128]. The first zone consists of mid-substance continuity of the enthesis is an efficient way to
or tendon proper; its composition has been transfer and buffer loads applied between mus-
described throughout the chapter. Basically, cle, tendon, and bone or from bone to bone. The
this zone contains collagen I-rich fibers that structure and composition of the region from
are aligned parallel to one another along the mid-substance to fibrocartilage accommodates
axis of strain, as well as a small amount of col- loads along the axis of the tendon, while the
lagens V, VI, XII, and XIV, decorin, and other region with mineralized fibrocartilage and bone
matrix macromolecules. The second zone is best zones manages complex multidirectional forces
characterized as fibrocartilage. It is predomi- that occur nearer to the bone [130]. The gradation
nantly composed of collagens II and III with of this transitional structure is difficult to repli-
minor amounts of collagens I, IX, and X, as well cate in surgery with native of engineered grafts.
24 M.J. Mienaltowski and D.E. Birk

2.17 Summary References

The functions of tendons and ligaments depend 1. Frank CBS, Shrive CB, Frank IKY, Hart DA (2007)
Form and function of tendon and ligament. In: Einhorn
greatly upon their extracellular matrices, par-
TA, O’Keefe RJ, Buckwalter JA (eds) Orthopaedic
ticularly the collagen that comprises 70–80 % basic science, 3rd edn. American Academy of
of their dry weight. Collagens found within Orthopaedic Surgeons, Rosemont, pp 199–222
tendons and ligaments belong to several subfami- 2. Birk DE, Bruckner P (2005) Collagen suprastruc-
tures. Curr Top Chem 247:185–205
lies that can be grouped by their predominant
3. Franchi M, Trire A, Quaranta M, Orsini E, Ottani V
suprastructural forms; these include: fibril- (2007) Collagen structure of tendon relates to func-
forming, FACIT, basement membrane, and tion. Sci World J 7:404–420
beaded filament-forming collagens. The many 4. Boot-Handford RP, Tuckwell DS (2003) Fibrillar
collagen: the key to vertebrate evolution? A tale of
collagens provide considerable diversity for
molecular incest. Bioessays 25(2):142–151
the functional extracellular matrix suprastruc- 5. Birk DE, Mayne R (1997) Localization of collagen
tures. This diversity is further compounded by types I, III and V during tendon development.
the numbers of different alpha chains formed; Changes in collagen types I and III are correlated
with changes in fibril diameter. Eur J Cell Biol
by alternative splicing; as well as by the many
72(4):352–361
types of post-translational modifications. 6. Riechert K, Labs K, Lindenhayn K, Sinha P (2001)
Overall it is the suprastructural organization of Semiquantitative analysis of types I and III collagen
these collagen molecules that provides tissue- from tendons and ligaments in a rabbit model. J
Orthop Sci 6(1):68–74
specific structure and function, particularly to
7. Fukuta S, Oyama M, Kavalkovich K, Fu FH,
tendons and ligaments. These suprastructures Niyibizi C (1998) Identification of types II, IX and X
are macromolecular heteropolymers containing collagens at the insertion site of the bovine Achilles
different collagens and other fibril-associated tendon. Matrix Biol 17(1):65–73
8. Milz S, Jakob J, Buttner A, Tischer T, Putz R,
molecules. Within tendons and ligaments, the
Benjamin M (2008) The structure of the coracoacro-
assembly of these collagen suprastructures mial ligament: fibrocartilage differentiation does not
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