− mouse model was found to result in the loss of most tendons and ligaments (Pryce et al., 2009) (Figure 1 G,H).
Growth and differentiation factors (GDF), members of the bone morphogenetic protein (BMP) family, are additional regulators of tendon development. Subcutaneous implantation of GDF-5, 6, and 7 leads to the formation of neotendon-like connective tissue in rats (Wolfman et al., 1997). Mice deficient in GDF-5 demonstrate inferior matrix composition and mechanical strength in their Achilles tendons (Mikic et al., 2001). Similarly, a null mutation of GDF-6 in mice is associated with substantially lower levels of tail tendon collagen content, which causes impaired mechanical integrity of the tissue (Mikic et al., 2009). However, GDF-7 deficiency does not affect the biochemical composition of mouse tail tendon fascicles, nor does it significantly affect the tensile material properties, suggesting differential effects of GDF isoforms in promoting tendon development (Mikic et al., 2008).
Upon aggregation, tendon progenitors of the developing embryo lay down small diameter collagenous fibrils (Banos et al., 2008; Connizzo et al., 2013), a process which continues after birth, with the fibrils growing in both the linear and lateral directions (Birk et al., 1995; Zhang et al., 2005). Fibrillar collagens, including types I, II, III, V, and XI, constitute the basic structural framework of tendons and ligaments. These collagens share a 300 nm-long triple helical domain comprised of three α chains, each containing about 1,000 amino acid residues. Each chain is rich in glycine and proline. As the smallest amino acid, glycine allows the three helical α chains to pack tightly together, while proline stabilizes the conformation due to its ring structure. Fibrillar collagens are synthesized in a precursor form, procollagen, that possesses a C- and N-terminal propeptide domain. Once secreted into the extracellular space, the propeptide domains are cleaved by specific telopeptidases. Mature fibrillar collagens generated from this modification spontaneously self-assemble into cross-striated fibrils in an entropy-driven manner, yielding a 67 nm repeat that characterizes the axial periodicity of collagen fibrils (Kadler et al., 1996). Collagen fibrils, 100-500 nm in diameter, are bundled into fibers between which tenocytes reside and maintain the extracellular matrix (ECM). The number and diameter of collagen fibers are highly variable among species, lower than 30 μm in rat tail tendon while higher than 300 μm in human tendons (Franchi et al., 2007). The collagen fibers are wrapped by a layer of connective tissue known as endotenon that contains blood vessels, lymphatics, and nerves, to form higher structural units called fascicles, which are surrounded by another connective tissue layer, epitenon, to form the tendon (Amiel et al., 1984; Kastelic et al., 1978) (Figure 2). Many of the tendons are further surrounded by loose areolar connective tissue called paratenon, which functions as an elastic sleeve and permits free movement of the tendon against the surrounding tissues.
Collagen type I is the predominant fibril-forming collagen in tendons and exists as a heterotrimer consisting of two α1 chains and one α2 chain. Collagen type I co-polymerizes with collagen type V, a known regulator of collagen fibrillar structure (Wenstrup et al., 2004). Non-fibrillar collagens, including the fibril-associated collagens with interrupted triple helices (FACIT) such as collagens type XII and XIV, also serve a regulatory role
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during collagen fibrillogenesis (Ansorge et al., 2009). Collagen type XII has been postulated to integrate adjacent matrix components due to its ability to bind proteoglycans, fibromodulin, and decorin, while interacting with collagen type I fibrils (Zhang et al., 2005). Collagen type XIV integrates fibrils into fibers during development, while collagen type XII assumes the same structural and functional role in mature tendon (Connizzo et al., 2013).
Expression and deposition of small leucine-rich proteoglycans, including decorin, biglycan, fibromodulin, and lumican, also serves to organize fibril assembly and the resulting ultrastructure of the tendon. Decorin-deficient mice developed structurally impaired tendons with abnormal, irregular fibril contours. Moreover, biglycan expression increased dramatically in decorin-deficient mice, suggesting a functional compensation (Zhang et al., 2006). Fibromodulin deficiency alone leads to a significant reduction in tendon stiffness, with further loss in stiffness when combined with lumican deficiency. Fibromodulin might be required to stabilize small-diameter fibrils in early tendon development, with lumican assuming this role later (Chakravarti, 2002). In addition to their regulatory effect on fibrillogenesis, biglycan and fibromodulin were also found to maintain the niche of tendon stem cells. The expression of the tendon marker Scx and of collagen type I was decreased in tendon stem cells from biglycan- and fibromodulin-deficient mice, as compared to cells from wild-type mice (Bi et al., 2007). Glycoproteins are also important constituents of tendon. Tenomodulin (TNMD), the type II transmembrane glycoprotein identified as a marker of mature tenocytes, is positively regulated by Scleraxis. In fact, TNMD is among the most tendon-selective genes in both adult rat and human tendons, compared with other tissues examined (Jelinsky et al., 2010). Loss of Tenomodulin expression in gene-targeted mice abated tenocyte proliferation and led to a reduced tenocyte density. The diameters of collagen fibrils varied significantly and exhibited increased caliber (Docheva et al., 2005). Tenascin C (TNC), member of a family of four ECM glycoproteins in vertebrates, was employed as the primary tendon marker prior to the discovery of Scx (Shukunami et al., 2006). It is an ECM component directly regulated by mechanical stress; induction of its mRNA in stretched fibroblasts is rapid, both in vivo and in vitro (Chiquet et al., 2003). Congruent with these findings of gene expression, the degree of tenascin C deposition is greatest in areas of high mechanical loading (Chiquet-Ehrismann and Tucker, 2004).
As tendons mature, covalent cross-links are formed between collagen fibrils at the overlapping ends of adjacent collagen molecules (Fessel et al., 2012) Marturano et al. (2013) recently showed that collagen cross-links correlate significantly with the increasing elastic modulus of in utero tendon, whereas correlations between mechanical properties and collagen, glycosaminoglycan, and dsDNA content were all weak (Marturano et al., 2013). The formation of collagen type I cross-links in tendons is primarily driven by the enzyme lysyl oxidase, which acts on specific lysine and hydroxylysine residues and results in stable trivalent cross-links that enhance collagen interconnectivity and fibril stability (Bailey, 2001; Eyre et al., 2008). While mechanical properties, collagen cross-links, average fibril diameter, and the distribution of fibril diameter, have all been found to increase with age, the structure-function relationship between these biochemical characteristics and tendon mechanical properties remains inconclusive (Connizzo et al., 2013