**3. Connective tissue elements in the dermis: Collagen fiber, elastic fiber, and ground substance**

Viscoelastic Properties of the Human Dermis and

Other Connective Tissues and Its Relevance to Tissue Aging and Aging–Related Disease 159

**Figure 1.** Immunohistochemical staining of fibrillin-1 in the human dermis. The distinct alignment of

Proteoglycans are glycosaminoglycans (GAGs) that are covalently linked to a core protein. GAGs can be chondrotin/dermatan sulfate, heparan sulfate, heparin, and hyaluronan. GAGs hold a large amount of water within connective tissue, whereas free water in connective tissue is observed as edema. In particular, hyaluronan (HA) has a high affinity to water through its charge and, in general, is a high molecular weight linear GAG that distributes ubiquitously in connective tissue. Proteoglycans are major components of ground substance and are occasionally associated with fiber components in the dermis. Therefore, GAG is essential for maintaining the tissue viscosity of dermal connective tissue. In dermal connective tissues, decorin and versican are the major proteoglycans (8). Decorin is a small dermatan sulfate proteoglycan that binds to type I collagen. In the dermis, decorin is abundant in the papillary and the reticular dermis. Scott et al. have proposed that, through their charge, GAG chains of decorin can play a role in the viscoelastic property of connective tissue (9, 10). These models highlight the importance of GAG chains to the viscoelastic

The supramolecular organization of the ECM in the dermis has been investigated using biochemical, biophysical, and ultrastructural methods. Connective tissues are not composed of a simple mixture of ECM molecules; therefore, the manner in which the ECM molecules assemble into fibrous components should be further investigated (11). In the dermis, each ECM molecule assembles into either elastic fiberous elements or ground substance. This can be observed in the electron micrograph of dermal connective tissue shown in Figure 2. Collagen and elastic fiber distribute distinctly, whereas the "empty space" is believed to be filled by ground substance. Thus, collagen and elastic fiber is embedded within ground substance, which itself is comprised of proteoglycans and

elastic fiber elements differs between the layers of the dermis

properties of connective tissues.

hyaluronan.

As the dermis is the layer that protects the body from physical stress, understanding the nature of dermal connective tissues is vital. In this section, 3 components of connective tissues and the ECM are briefly introduced. ECM molecules are produced primarily by fibroblast cells.

Collagen fibers are major elements of the dermis and collagens are the most abundant protein in the human body; the dermis alone is composed of approximately 75% collagen proteins in dry weight. Twenty-eight collagen species have presently been identified. It has been reported that skin contains collagen types I, III, IV-VII, XIII, and XIV, with the major collagen in the dermis being type I collagen. Collagens that associate with the type I collagen fiber are classified as FACIT collagens and can provide additional mechanical properties to tissues. Collagens are characterized by repeated glycine-X-Y sequences and form triplehelical structures that are extensively modified after their secretion into the extracellular space. In immature tissues, such as those found in wound healing and fibrosis, type III collagen is expressed; however, it is not yet strong enough to support mature connective tissues. As the wound matures, type I collagen becomes dominant. Heterotypic type I and type III collagen fibrils are present in the dermis. Type VI collagen individually forms a unique filament called a microfilament (1,2).

Elastic fiber comprises elastin and microfibrils. As the dermis has to be stretched to adapt to the movement of body parts, elasticity is a critical property of the dermis. Elastin—a unique molecule that stretches and shrinks—is secreted as tropoelastin (the soluble precursor of mature elastin) and is subsequently processed and cross-linked within the extracellular space. Cross-linking by lysyl oxidase and desmosine formation is a crucial step for the stabilization of elastin within tissues. Another element in elastic fibers is fibrillin-microfibril. Microfibrils are fibrous elements that are 10 nm in width and are comprised mainly of fibrillins. Fibrillin is a large glycoprotein that is rich in cysteine residues and homotypically assembles into a microfibril in a well-regulated manner (3, 4). Fibrillins align in a parallel manner, from head to tail, in a staggered fashion within extracellular microfibrils (5). Other ECM molecules, including microfibril-associated glycoproteins (MAGPs), latent TGF-beta binding proteins (LTBPs), type XVI collagen, emilin, and versican, can associate with microfibrils through their binding affinity with fibrillins. Fibulins are yet another elastic fiber component, which can bridge elastin and microfibrils by their binding properties. Interestingly, fibulin-5 knock-out mice exhibit skin looseness (6, 7), indicating that this molecule may be essential for the development of elastic tissue.

Thick elastic fiber distributes horizontally in the reticular dermis, whereas thinner elastic fiber, including elaunin and oxytalan fibers, are seen to distribute in the papillary dermis. Oxytalan fibers are formed by bundled microfibrils without amorphous elastin. The staining of fibrillin in the skin shows horizontal distribution in the reticular dermis and vertical orientation in the papillary dermis (Figure 1). This complex elastic fiber meshwork confers the dermis with the ideal viscoelasticity to effectively protect the human body.

unique filament called a microfilament (1,2).

molecule may be essential for the development of elastic tissue.

**and ground substance** 

fibroblast cells.

**3. Connective tissue elements in the dermis: Collagen fiber, elastic fiber,** 

As the dermis is the layer that protects the body from physical stress, understanding the nature of dermal connective tissues is vital. In this section, 3 components of connective tissues and the ECM are briefly introduced. ECM molecules are produced primarily by

Collagen fibers are major elements of the dermis and collagens are the most abundant protein in the human body; the dermis alone is composed of approximately 75% collagen proteins in dry weight. Twenty-eight collagen species have presently been identified. It has been reported that skin contains collagen types I, III, IV-VII, XIII, and XIV, with the major collagen in the dermis being type I collagen. Collagens that associate with the type I collagen fiber are classified as FACIT collagens and can provide additional mechanical properties to tissues. Collagens are characterized by repeated glycine-X-Y sequences and form triplehelical structures that are extensively modified after their secretion into the extracellular space. In immature tissues, such as those found in wound healing and fibrosis, type III collagen is expressed; however, it is not yet strong enough to support mature connective tissues. As the wound matures, type I collagen becomes dominant. Heterotypic type I and type III collagen fibrils are present in the dermis. Type VI collagen individually forms a

Elastic fiber comprises elastin and microfibrils. As the dermis has to be stretched to adapt to the movement of body parts, elasticity is a critical property of the dermis. Elastin—a unique molecule that stretches and shrinks—is secreted as tropoelastin (the soluble precursor of mature elastin) and is subsequently processed and cross-linked within the extracellular space. Cross-linking by lysyl oxidase and desmosine formation is a crucial step for the stabilization of elastin within tissues. Another element in elastic fibers is fibrillin-microfibril. Microfibrils are fibrous elements that are 10 nm in width and are comprised mainly of fibrillins. Fibrillin is a large glycoprotein that is rich in cysteine residues and homotypically assembles into a microfibril in a well-regulated manner (3, 4). Fibrillins align in a parallel manner, from head to tail, in a staggered fashion within extracellular microfibrils (5). Other ECM molecules, including microfibril-associated glycoproteins (MAGPs), latent TGF-beta binding proteins (LTBPs), type XVI collagen, emilin, and versican, can associate with microfibrils through their binding affinity with fibrillins. Fibulins are yet another elastic fiber component, which can bridge elastin and microfibrils by their binding properties. Interestingly, fibulin-5 knock-out mice exhibit skin looseness (6, 7), indicating that this

Thick elastic fiber distributes horizontally in the reticular dermis, whereas thinner elastic fiber, including elaunin and oxytalan fibers, are seen to distribute in the papillary dermis. Oxytalan fibers are formed by bundled microfibrils without amorphous elastin. The staining of fibrillin in the skin shows horizontal distribution in the reticular dermis and vertical orientation in the papillary dermis (Figure 1). This complex elastic fiber meshwork confers

the dermis with the ideal viscoelasticity to effectively protect the human body.

**Figure 1.** Immunohistochemical staining of fibrillin-1 in the human dermis. The distinct alignment of elastic fiber elements differs between the layers of the dermis

Proteoglycans are glycosaminoglycans (GAGs) that are covalently linked to a core protein. GAGs can be chondrotin/dermatan sulfate, heparan sulfate, heparin, and hyaluronan. GAGs hold a large amount of water within connective tissue, whereas free water in connective tissue is observed as edema. In particular, hyaluronan (HA) has a high affinity to water through its charge and, in general, is a high molecular weight linear GAG that distributes ubiquitously in connective tissue. Proteoglycans are major components of ground substance and are occasionally associated with fiber components in the dermis. Therefore, GAG is essential for maintaining the tissue viscosity of dermal connective tissue. In dermal connective tissues, decorin and versican are the major proteoglycans (8). Decorin is a small dermatan sulfate proteoglycan that binds to type I collagen. In the dermis, decorin is abundant in the papillary and the reticular dermis. Scott et al. have proposed that, through their charge, GAG chains of decorin can play a role in the viscoelastic property of connective tissue (9, 10). These models highlight the importance of GAG chains to the viscoelastic properties of connective tissues.

The supramolecular organization of the ECM in the dermis has been investigated using biochemical, biophysical, and ultrastructural methods. Connective tissues are not composed of a simple mixture of ECM molecules; therefore, the manner in which the ECM molecules assemble into fibrous components should be further investigated (11). In the dermis, each ECM molecule assembles into either elastic fiberous elements or ground substance. This can be observed in the electron micrograph of dermal connective tissue shown in Figure 2. Collagen and elastic fiber distribute distinctly, whereas the "empty space" is believed to be filled by ground substance. Thus, collagen and elastic fiber is embedded within ground substance, which itself is comprised of proteoglycans and hyaluronan.

Viscoelastic Properties of the Human Dermis and

Other Connective Tissues and Its Relevance to Tissue Aging and Aging–Related Disease 161

chains that hold a large amount of water within the ECM space; and 3) binds to HA, which holds a large quantity of water. Figure 4 shows the proposed structural model of the elastichydrated matrix in the dermis. Furthermore, the fibrillin-versican-hyaluronan network is

**Figure 3.** Versican co-localizes with fibrillin-1 in the dermis. Immunofluorescent staining using specific

**Figure 4.** Schematic presentation of the dermal viscoelastic network linked by versican. Versican links

antibodies against versican and fibrillin-1 show co-localization.

elastic fibers to ground substance.

also observed in the ciliary body (16).

**Figure 2.** Electron microscopic image of dermal connective tissues. Col: collagen fiber, El: elastin core, MF: microfibrils (Bar = 200 nm)
