Materials Science and Mechanics of Collagenous Tissues

Abstract

Collagen is the major structural protein found in mammalian extracellular matrix (ECM). ECMs act as biological mechanotransducers that prevent premature mechanical failure of tissues, store and transmit energy created by muscular deformation, and amplify protein synthesis and cell division as the applied stress and loads are increased (mechanochemical transduction). Fibrous collagens play important roles in health and disease processes much of which depends on the mechanical properties of these tissues. The purpose of this paper is to summarize our knowledge of relationship between the structure and mechanical properties of fibrous collagens in vertebrates.

Keywords

Collagen, Viscoelasticity, Mechanical properties, Modulus, Skin Tendon, Bone

Introduction

Collagen is the most abundant protein found in the extracellular matrix (ECM) of vertebrates. It forms the fibrous backbone or parenchyma of many tissues, the support for cells at tissue interfaces, and is the primary structural component of the musculoskeletal system. The collagen family is composed of several collagen subfamilies including fibril forming collagens, beaded filaments, anchoring fibrils and network forming collagens [1]. The structural stability of tissues is the result of the formation of collagen fibrils, fibril bundles, fibers and fascicles in tendon, and in other tissues collagen fibrils and fibers are the structural components. Most collagen fibrils are composites since they are made up of more than one collagen types such as tendon (types I, II and V), cartilage (types II, IX and XI), skin (types I and III) and cornea (types I, III and V). In addition, other non-collagenous macromolecules are attached to collagen fibrils that are involved in fibril formation (proteoglycans), cell-collagen interactions (fibronectin) and mechanotransduction (integrins) [1]. The purpose of this paper is to relate the structural hierarchy of collagen fibrils and fibers to the mechanical behavior of collagenous tissues to help better understand the function of collagen in health and disease.

Mechanical and Metabolic Roles of Collagen in Tissues

Collagen fibers in vertebrate extracellular matrix (ECM) serve important mechanical roles including preventing premature mechanical failure and modulation of force transfer between neighboring tissues [2,3]. They store elastic energy during muscular deformation, transmit stored energy resulting in joint movement, transfer excess energy from the joint back to the attached muscles for dissipation and promote regulation of cell and tissue synthesis through a process known as mechanochemical transduction [4]. They act as mechanotransducers by transferring stress borne by the musculoskeleton and other tissues to the attached cells in order and regulate tissue metabolism, either up- or down, as a result of changes in mechanical loading [3]. Collagen fibers transduce mechanical loading into changes in chemical synthesis of proteins which leads to energy storage in the form of high molecular weight proteins (mechanochemical transduction). Therefore, collagen fibril and fiber structure is intimately related to: energy storage, transmission, dissipation, premature mechanical failure of tissues, and up- and down-regulation of tissue metabolism. Changes in collagen structure have been noted in diseases such as cancer, scarring, osteoarthritis and inflammation suggesting that collagen structural changes may be a useful parameter to follow in the early diagnosis of these diseases.

Location and Function of Collagenous Tissues in Vertebrates

Collagen fibrils and fibers are found in a variety of tissues throughout the body. They can be found in both unmineralized and mineralized tissues and can be classified into the following categories: (1) surface and internal lining tissues, (2) conduit and holding structures, (3) parenchymal or organ-supporting tissues, and (4) dental and musculoskeletal tissues [5].

The ECM of surface and internal linings is composed of collagen, proteoglycans, elastin, glycoproteins, cells and water and is found in internal and external lining structures at the interfaces with the air or internal tissue [5]. Examples of these include cornea, skin, alveoli, oral mucosa, vagina, and uterus. These sheet-like tissues provide mechanical, chemical, and microbiological barriers to keep what is in the environment from penetrating into host tissue (Figure 1). The internal linings such as pleural and peritoneal membranes protect organs in the thoracic and abdominal cavities from injury. Collagen fibrils and fibers in this class of tissues are found as networks that can be considered planar reinforcement elements that provide mechanical support but they also are involved in mechanotransduction as described below [3]. Skin and cornea are examples of surface and internal lining structures and contain surface cellular layers that cover collagenous supporting layers.

A second classification of tissues includes conduit and holding structures, which are tubes and containers with three or more layers that make up the walls. For example, blood vessels (Figure 2) contain an intima or cellular layer in contact with the flowing blood, a muscular or medial layer, and an outer layer termed adventitia that blends with the surrounding tissues. Other tubular structures are similar in that they consist of an internal cellular layer (mucosa), surrounded by a muscular layer (muscularis), and an external ECM containing layer (serosa). Collagen fibrils and fibers in these tissues are mixed with elastic tissue and smooth muscle cells and serve to reinforce the tissue walls [5].

Parenchyma and organ supporting collagens form the scaffold of major organs such as liver, kidney, heart and lymph nodes [5]. The collagen fibrils and fibers in these tissues support cells and in conjunction with other tissue elements allow cellbased physiologic processes to occur within a fixed organization of cells. In liver, the collagen fibrils support the endothelial cells that line the sinusoids allowing large quantities of blood to be filtered.

The last classification of tissues is dental and musculoskeletal tissues. These tissues include anterior cruciate and other ligaments, spongy and dense long bones, periodontal ligament, tendons, and intervertebral disc of the back [5]. The collagen fibrils and fibers of these tissues provide structural stability in the presence and absence of mineral termed hydroxyapaptite (Figure 3).

Thus fibrous collagen is found in almost every tissue and organ in vertebrates. Collagen fibers are composed of collagen triple-helical molecules that are derived from one or more of the 28 collagen types that compose the collagen family [1]. While we will concentrate on fibrous collagens in this paper, the collagens of the other subfamilies plays additional roles in mammalian tissue structure and function.

Collagen Molecular Structure

All collagens contain amino acid sequences of gly-X-Y where X and Y are frequently proline and hydroxyproline. The sequence gly-X-Y leads to the formation of a left-handed triple helix and three left handed triple-helices form a right handed superhelix. The length of the triple helical segments varies between collagen sub-families with the longest triple-helical segments found in the fibril forming collagens that have about 1000 amino acids in a single left handed helix.

In the fiber forming collagens, this three chained molecule forms a right-handed triple helical structure containing glycine residues buried at the center of the cylindrical molecule and is synthesized in a longer precursor form termed procollagen (Figure 4). The most abundant subfamily is the fibrillar collagens of which type I collagen is found in tendons, skin, cornea, bone lung and vessel walls [6]. This collagen is thought to give rise to the high tensile strength of collagen fibers in tendon; in addition, it actively is involved

Collagen synthesis from amino acids begins on the ribosomes within the cell cytoplasm; the synthesized chain is then folded into a triple helix after hydroxylation of proline is completed. The molecules are then transported through the Golgi apparatus, packaged into vesicles, and then are added to growing fibrils in recesses within the cell membrane. However, collagen fibrils are not mechanically stabilized until they are crosslinked which occurs after release from the cell and mechanical loading begins [7,8].

Procollagen triple helices contain extra pieces on the C and N terminal ends of the molecule termed propeptides (Figure 4). The propeptides are removed sequentially during collagen assembly. Collagen triple helices, 1.5 nm wide and 300 nm long, are packed into a “quarter-staggered” packing pattern that results in nearest neighboring molecules being staggered longitudinally by about 22% of their molecular lengths with a space or hole between the head of one molecule and the tail of the next (Figure 5). Early studies recognized that five collagen molecules were packed in a staggered fashion to form a microfibril [9,10] that was identified by electron microscopy [11]. It is now believed that collagen molecules are packed laterally into quarter-staggered quasi-hexagonal units that are in turn longitudinally packed into microfibrils. Collagen microfibrils are believed to be continuous and run the length of a tissue; they are fused laterally into fibrils and fibers in most tissues [7,8,12,13].

In the 1970s collagen molecule was thought to be a rigid rod based on the observation that the translational diffusion constant was 0.86 × 10^-6 cm2/sec, which was very close to that calculated for a prolate ellipsoid 1.5 nm wide and 300 nm long [14]. However, later measurements, based on rotary shadowed images of collagen molecules, suggested that the molecules had numerous bends as shown in Figure 6a [12]. Analysis of the molecular sequence of type I, II and III collagens suggested that the flexibility of the collagen triple helix arises from areas in the sequence that are devoid of proline and hyroxyproline, two imino acids that constrain rotation and flexibility of the molecule [13] (Figure 5). Further analysis suggested that these flexible regions are preserved when the molecules are packed into a quasi-hexagonal packing pattern indicating that flexibility is an important aspect of the collagen fibril structure [13].

Supramolecular Structure of Collagen

Collagen in tissues is recognized by transmission microscopy by its regular repeat of the charged amino acid residues. In the quarter-staggered packing pattern, the amino acid sequence of the five molecules in cross-section is repeated every 64 to 67 nm, a distance termed the D period (Figure 5). The positively stained sub-bands in the D period (see Figure 6) can be depicted as spring-like (see Figure 5) and can be directly visualized by staining with heavy metals (Figure 6). The D period varies from about 64 to 67 nm depending on the tissue of origin. In tendon, the D period is about 64 nm and in skin it is about 67 nm (Figures 6c-6e). At the light microscopic level, collagen fibers in tendon are distinguished from other tissue proteins by the cross striated pattern derived from the crimp seen under polarized light (Figure 6k). Other tissues contain collagen fibers in aligned (tendon) and oriented networks (skin) that are visualized under polarized light.

For instance in tendon, collagen fibril diameters are between 20 and 280 nm and collagen fibers diameters are between 1 and 300 μm [13]. While collagen fibrils form from lateral addition of smaller fibrils, they form bundles of fibrils and larger bundles termed fascicles as shown in Figure 3. Groups of fibril bundles form fascicles that in turn make up the cross-section of a tendon bundle (Figure 3). These structural elements acting in concert give rise to the mechanical properties of tendon and allow structures such as the finger joints to bend independently.

The ultrastructure of tendon has been studied extensively as a function of maturation. Collagen microfibrils appeared to be held together by an interfibrillar matrix containing proteoglycans. These collagen fibrils were observed to have a planar waveform, termed crimp, and be the load bearing units [15] (Figure 6k). Upon deformation, the stress-strain curve of tendon curves upward after the crimp is removed [15]. After birth, the distribution of fibril diameters from rat tail tendons is fairly flat supporting the concept that fibril diameter and length increases occur by the lateral fusion of fibril bundles [7,8,12,16,17]. The volume fraction of collagen fibrils increases during maturation until it reaches about 0.5; in fibril cross-sections of tendon small collagen fibrils fill the space between larger ones [16].

Collagen Crosslinking

The ability of fibril forming collagens to store, transmit and dissipate energy requires crosslink formation between molecules within a microfibril and between microfibrils and other structural units [3] as indicated in Figure 5. These crosslinks include lysine and hydroxylysine derivatives and other amino acid residues including histidine [3,18]. Crosslinking also occurs during aging and involves glucose molecules [1]. The stiffening and poor energy dissipation of collagen fibers associated with aging probably involves collagen fiber fragmentation by UV light and glucose derived crosslinking. This leads to loss of the energy dissipating ability of extracellular matrix and is associated with large energy losses during pulsatile blood flow in the cardiovascular system [19].

Collagen fibers are viscoelastic and exhibit time-dependent mechanical behavior (Figure 7). Viscoelasticity may be important in resisting impact loads at low strain-rates especially in the musculoskeleton, however, it complicates the understanding of ECM behavior since most real-time measurements made on these tissues contain both elastic and viscous contributions [2]. The elastic behavior varies from as high as about 75% of the total stress for tendon to as low as about 50% for skin depending on the collagen fiber orientation, rate of loading and the quantity of other tissue constituents [2]. Results of recent studies suggest that the elastic modulus of collagenous tissues can be measured by measuring the resonant frequency of a tissue using optical coherence tomography and vibrational analysis which can be done non-invasively and non-destructively [20,21].

Role of Collagen in Energy Storage, Transmission and Dissipation

Energy storage, transmission and dissipation are some of the many key mechanical functions provided by fiber forming collagens of the ECM found in vertebrate tissues. These functions are required for effective locomotion, tissue regeneration and repair through mechanotransduction processes [3]. The above mechanical functions are required for vertebrates to achieve locomotion and move efficiently. Vertebrates must be able to develop muscular forces, store elastic energy, and transfer this energy to the attached joints for locomotion to occur. In addition, excess energy remaining after movement is achieved must be transferred from the joint back to the muscles where it can be dissipated as heat [3]. It is important to examine at the molecular and tissue levels how energy storage, transmission and dissipation occur.

Mechanistically, during mechanical loading of tendon it has been shown that a tensional increase in the D period is observed with increasing strain that is associated with: (1) molecular elongation at the triple-helical level of structure; (2) increases in the gap distance between the end of one triple-helix and the start of the next one in the microfibril; and (3) molecular slippage [22]. Molecular stretching occurs at lower stresses followed by increases in the gap spacing (Figure 5) and molecular sliding; the latter occur at higher stresses [23].

A molecular modeling program has been used to calculate the change in free energy associated with a 1-3% increase in the h spacing of a type I collagen triple helix packed into a five-membered unit. The resulting free energy calculation was compared to the change in energy under the molecular stress versus strain curve. From these considerations it was determined that changes in free energy during stretching a collagen microfibril were proportional to the energy changes under the molecular stress-strain curve [24]. Furthermore, the regions without imino acids appear to “open up” or uncoil when stress is applied. They appear to serve as sites that can store energy elastically. Peterlini et al. [25] have predicted that these regions can form folds in the collagen triple helix; the folds may unfold when stress is applied and energy is stored. Molecular modeling results suggest that stretching increases steric energy of the triple helix that is attributable to van der Waals and electrostatic interactions between amino acids that are charged [24].

Mechanotransduction and Mechanochemical Transduction

ECMs modulate internal mechanical forces into changes in biochemical signals in a series of phosphorylation events in a process termed mechanotransduction [3]. If proteins and new high molecular weight tissue are formed this process becomes mechanochemical transduction. An example of this process may be described by lifting weights repeatedly for several months. In this situation both the force generated by the muscle increases (mechanotransduction) and the size of the muscles and the amount of skin increase (mechanochemical transduction) [3,13]. These changes occur because many of the ECMs in the human body are under internal tension. The need to balance external forces resulting from lifting weights and the internal forces exhibited by the collagen networks and the resident cells drives mechanochemical transduction [3]. Alteration of the balance between external and internal mechanical forces acting on resident cells found in ECMs, leads to changes in gene expression and subsequent production of ECM proteins [3]. In contrast to tissue stimulation by weight lifting, prolonged bed rest and a reduction in external mechanical loading results in tissue catabolism. This leads to loss of muscle and atrophy of the musculoskeletal system [3]. Therefore any changes in the equilibrium between external and internal mechanical forces acting on ECMs lead to changes in mechanochemical transduction at the cellular level. These changes appear to be important mechanisms by which mammals adjust their needs to store, transmit and dissipate energy that is required for bodily movements in a gravitational field [3].

Mechanochemical transduction is an important process that results in changes in ECM hierarchical structure in both health and disease. Therefore, the relationship between mechanical behavior and hierarchical structure must be considered in more detail. Figure 8 summarizes some of the steps that are involved in converting mechanical loading into changes in tissue structure.

Non-Invasive and Non-Destructive Methods to Determine the Mechanical Properties of Tissues

The ability to monitor the mechanical properties of collagen and ECMs in vivo is an important measurement needed for early diagnosis of disease and the ability to follow disease progression. For over 100 years physicians have been able to “palpate” changes in the properties of tissues associated with tumors and calcification; this suggests that there are major changes in the structure and properties of collagen and ECMs during disease processes. It is essential that clinicians be able to assess the changes at the collagen fibril and fiber levels of structure to accurately diagnose and treat diseases such as cancer and osteoarthritis. Several new methods have been developed to try and discern these changes early in the disease process. It is essential that these methods be validated so that the properties measured have some meaning.

We have reported [20,21] the use of vibrational analysis in concert with optical coherence tomography (OCT) to measure the resonant frequency and modulus of a number of tissues. The resonant frequency measured is directly related with the tensile modulus obtained from an incremental stress-strain experiment (Figure 9). The method was recently extended to measuring the modulus of normal skin and scar tissue in vivo [21].

Conclusions

Collagen is the major structural protein found in mammalian ECMs. It is found in a number of different forms and is important in the structural stability of tissues. ECMs act as biological mechanotransducers that prevent premature mechanical failure of tissues. ECMs store and transmit energy created by muscular deformation and amplify protein synthesis and cell division as the applied stress and loads are increased (mechanochemical transduction). They regulate their size and shape as a result of changing local mechanical demands to tissues. Changes in tissue metabolism are transduced into increases or decreases in synthesis and catabolism of the components of ECMs. In synthesizing high molecular weight macromolecules, energy is stored in the form of covalent bonds. This energy can be released by tissue catabolism if needed.

Energy storage and elastic behavior primarily involve the stretching of flexible regions devoid of imino acid residues within collagen triple helices found in crosslinked collagen fibrils. Since tissue architecture varies among different ECMs so too does the amount of energy that can be stored and dissipated.

It is now possible to evaluate structure-properties relationships for ECMs in vivo using OCT and vibrational analysis. This enables clinicians to “quantitatively palpate” tissues to elucidate the relationship between structure and properties in both health and disease.

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