Collagen is the major protein in ligaments, and it is not a single entity. It is the single most abundant animal protein in mammals, accounting for up to 30% of all proteins. The collagen molecules, after being secreted by the cells, assemble into characteristic fibers responsible for the functional integrity of tissue such as bone, cartilage, skin, ligaments, and tendon [8]. They contribute a structural framework to other tissues, such as blood vessels and most organs. Cross-links between adjacent molecules are a prerequisite for the collagen fibers to withstand the physical stresses to which they are exposed. Significant progress has been made toward understanding the functional groups on the molecules involved in the formation of such cross-links, their nature and location, etc. A variety of human conditions, normal and pathologic, relate to repair and regeneration of the collagenous framework of tissues. Some of these conditions are characterized by excessive deposition of collagen (e.g., cirrhosis, scleroderma, keloid, pulmonary fibrosis, diabetes). After trauma or surgery, abnormal deposition of collagen may impair function (adhesions following repair or scar formation during healing). In addition, many disabling conditions result from changes in the nature of collagen (heart valve lesions, osteoarthritis, rheumatoid arthritis, and congenital collagen diseases such as Marfan’s and Ehler–Danlos syndromes and osteogenesis imperfecta.

The arrangement of amino acids in the collagen molecules is shown schematically in Fig. 23.2. Every third amino acid is glycine. Proline and hydroxyproline follow each other relatively frequently, and the sequence (gly, pro, hyp) makes up about 10% of the molecule. This triple helical structure generates a symmetrical pattern of three left-handed helical chains that are, in turn, slightly displaced to the right, superimposing an additional “supercoil” with a pitch of approximately 8.6 nanometers (nm). These chains, known as alpha chains, have a molecular weight of around 100 kDa and contain approximately 1000 amino acids for the interstitial collagen Types I, II, and III (Fig. 23.3). The amino acids within each chain are displaced by a distance of h = 0.201 nm, with a relative twist of 100°, making the number of residues per turn 3.27, and the distance between each third glycine 0.87 nm.

The individual residues are nearly fully extended in the collagen structure, as the maximum displacement within a fully stretched chain would be approximately 0.36 nm. This separation will not allow interchain bonds to form, and only interchain hydrogen bonds are possible. In addition to these intramolecular conformational patterns, there appears to be a supermolecular coiling. A process of self-assembly causes the collagen molecules to organize into fibers.

Development of an extracellular network of collagen fibers requires cells involved in biosynthesis to synthesize procollagen. This molecule is then enzymatically trimmed of its nonhelical ends, and the resultant collagen molecule assembles into fibers in the extracellular space. Procollagen molecules have been identified as precursors of three interstitial collagens (Types I, II, and III). Several of the N-(amino) and C-(carboxy) terminal peptides (propeptides) have been characterized and the primary sequences determined.

The carboxyterminal propeptides of both α₁ and α₂ chains have molecular weights of 30,000 to 35,000 Da, and globular conformations without any collagen-like domain. These peptides contain asparagine-linked oligosaccharide units composed of N-acetylglucosamine and mannose. Once the molecule is completed and translocated to the cell surface, the extensions are enzymatically removed from those collagens, which then form fibrils. Enzymes that selectively remove these extensions can be found in a variety of connective tissues and in the culture media derived from collagen-secreting cells.

Over the past 40 years since the discovery of type II collagen in cartilage, many other species of collagen have been identified. Types I, II, III, V, and XI collagen are categorized as fiber-forming collagens. They all exhibit lengthy, uninterrupted collagenous domains and are first synthesized as biosynthetic precursors (procollagens). Gene cloning experiments have demonstrated that Group I collagen genes are evolutionarily related, for they share a common ancestral gene structure. Human chromosome number 17 contains the coding information for the α₁ chain of Type I collagen, while chromosome 7 codes for its complementary α₂ chain. A comparison of the five fibrillary collagens described shows that, with one exception, (Types III and α₂ (V) are located on chromosome 2), all other genes are located on different chromosomes.

The gene codings for fiber-forming collagens are large, about 10 times the size of the functional mRNA. Many of the exons (coding sequences) are 54 base pairs (bp) in length, and are separated from each other by large intervening sequences (introns) that range in size from ~80 to 2000 bp. The gene itself contains 38,000 bp and is very complex. The finding that most exons of these genes have identical lengths suggests that the ancestral gene for collagen was assembled by multiple duplications of single genetic units containing an exon of 54 bp (Fig. 23.4).

As this chapter focuses on ligaments, the major characteristics of the two collagen types present in the ACL, namely Types I and III, are described. Type V collagen seems to be present at less than 1%.

Proteoglycans consist of small amounts of protein bound to negatively charged polysaccharide chains referred to as GAGs. In articular cartilage, proteoglycans form a large portion of the macromolecular framework (commonly about 30–35% of the tissue dry weight), but in ligaments they form only a small portion of this framework, usually less than 1% of the dry weight [13, 18, 19]. Nonetheless, proteoglycans may have an important role in organizing the extracellular matrix and interacting with the tissue fluid [20–27].

Like tendon, meniscus, and articular cartilage, ligaments contain two known classes of proteoglycans: large articular-type proteoglycans containing long, negatively charged chains of chondroitin and keratin sulfate (syndecan) and smaller proteoglycans that contain dermatan sulfate [22–24]. Because of their long chains of negative charges, the articular cartilage-type proteoglycans tend to expand to their maximum domain until restrained by the collagen fibril network. As a result, they maintain water within the tissue, alter fluid flow within the tissue during loading, and exert a swelling pressure, thereby contributing to the mechanical properties of the tissue and filling the region between collagen fibrils.

These molecules are composed primarily of protein, but many of them also contain a few monosaccharides and oligosaccharides [19, 22, 24]. Although noncollagenous proteins such as fibronectin contribute only a few percentage points to the dry weight of ligaments, they have an important role in the complex interaction of ligament cells and their environment during growth, healing, and remodeling. This role, however, is poorly understood.

Fibronectins are important in an array of cellular functions, particularly those involving a cell’s interaction with its surrounding extracellular matrix. They are high-molecular-weight extracellular glycoproteins whose functions include modulating intra- and extracellular matrix morphology, cellular adhesion (both cell-to-cell and cell-to-substratum), and cell migration. Examined by electron microscopy, fibronectins appear as fine filaments or granules coating the surface of fibrillary collagens or associated with cell membranes. Fibronectins have an adhesive domain specific to fibrin, actin, hyaluronic acid, cell surface factors, and collagen. They function to attract and couple key elements in normal healing and in growing tissue.

Quantitative studies of fibronectin concentrations in rabbit ligaments demonstrate significantly (two to three times) higher amounts of fibronectin in the cruciate ligaments as compared to the collateral ligaments [13]. This difference may reflect the fact that the cruciate ligaments are surrounded by a synovial sheath, and therefore have a higher degree of cellularity relative to the extraarticular ligaments.

The maintenance of ligament tissue and its ability to respond to load changes depend on interactions between the cells and matrix. Normally, the matrix macromolecules are slowly but continually degraded and replaced. The cells must synthesize new macromolecules to balance the losses due to normal degradation or microtrauma. The matrix provides to the cells protection from mechanical injury during normal loading and transmits signals generated by loading to the cells.

Cells bind to the matrix primarily through a family of cell surface proteins called integrins. These molecules mechanically link the matrix macromolecules, including fibronectin, to the internal cell cytoskeleton. They participate in cell adhesion, migration, and proliferation, and in regulation of cell synthesis of new matrix macromolecules (Fig. 23.5).

A vast and rapidly growing amount of literature abounds on a class of peptides commonly called growth factors. Accelerated healing of skin wounds has been reported after local application of several growth factors [28–30].

After injury the platelets travel to the wound site, form a clot, and hemostasis is obtained. Platelets secrete peptides such as platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β). Both PDGF and TGF-β play an important role in the initiation of repair processes after injury. These factors are chemotoxic for inflammatory cells, and appear to regulate proliferation and differentiation of fibroblasts [31–36]. Inflammatory cells at the wound site then release other peptides such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). bFGF has demonstrated stimulatory effects on angiogenesis, urokinase-type plasminogen activator (implicated in the neovascular response), and wound healing [37]. PDGF has demonstrated beneficial effects upon ligament healing upon testing in a medial collateral ligament injury model in rats [38]. The administration of platelet-rich plasma has been shown to enhance collagen remodelingand hypercellularity with increased metabolic activity during the healing process [39]. Collagen PRP hydrogel has been shown to improve healing of the ACL with improved wound site healing in a canine injury model [40]. VEGF has been shown to increase healing via neogenesis [41, 42].

Because synovial fluid washes clots away from the ligament injury site, it is hypothesized that a deficiency of growth factors exists at the wound site. Without the necessary stimulus from growth factors and other clot-derived substances, the response to injury is poor.

Lyon et al. [43] further revealed cellular alignment differences. The deep portion of the central one-third of the MCL had compact collagen fiber bundles oriented along the longitudinal axis of the ligament. Spindle-shaped MCL fibroblasts were interspersed throughout the collagen fiber bundles. Transillumination of the MCL using polarized light demonstrated a high-amplitude, low frequency crimp pattern of the collagen fibers, which parallels that of adjacent fiber bundles. MCL fibroblasts were oriented at angles corresponding to the angle of collagen fiber crimp (Fig. 23.6a). The deep portion of the central third of the ACL also had compact, parallel collagen fiber bundles oriented along the long axis of the ligament. However, the fiber bundles were separated by narrow spaces containing ovoid cells arranged in columns like pearls on a string. The crimp pattern for the ACL collagen fiber bundles demonstrated a low amplitude, high frequency pattern (Fig. 23.6b). Furthermore, the cells within the narrow spaces did not conform to the crimp pattern of the adjacent fibers. The anteromedial bundle of human ACLs distinguished three different zones along the length of the ligament. These zones were characterized by fusiform, ovoid, and spheroid cell shapes. Fusiform and ovoid cells occupied the proximal quarter of the anteromedial bundle of the ACL and were found to express the alpha smooth muscle actin isoform. In the spheroid zone, which constitutes the distal three fourths of the ACL, only a portion of cells expressed the alpha smooth muscle actin isoform [44]. These cellular alignment differences may represent a spectrum among ligamentous cells with the classic fibroblast-fibrocyte at one end and the chondroblast-chondrocyte at the other end [44]. But in general, the MCL histologically appears to favor the fibroblast-fibrocyte phenotype, whereas the ACL cell tends to favor a fibrocartilage phenotype [43].

Cellular and biologic processes need to be discussed to understand the responses to ligament injury. The cellular difference in the ACL versus the MCL includes differences in phenotype, cellular alignment, proliferation, migration, adhesion to substrates, responses to mechanical forces, and signaling. Nagineni et al. [45] studied knee ligament histology and found that MCL cells exhibited a typical fibroblastic morphology. The cells were elongated and spindle shaped. ACL cells, however, were slightly larger and more ovoid in shape. Lyon et al. [43] in our laboratory reported that ACL cells had a more fibrocartilage characteristic.

Cellular cytoplasmic processes were found to be different. The MCL fibroblast has long cytoplasmic processes extending outward into the surrounding matrix. In striking contrast, the ACL cells are devoid of any long cytoplasmic processes, and the cell membrane and the adjacent collagen fibrils are separated by an amorphous matrix [43]. When these cells were stained for fibronectin, both cell types showed intense staining in the area of the cell membrane. The major difference was that the fibronectin stain followed the MCL processes far out into the matrix. Because the ACL cells lack the long processes, they did not have the same staining pattern [43] (Fig. 23.7). Burridge and Chrzanowska-Wodnicka [46] found more bundles of microfilaments (representing stress fibers) in the ACL than in the MCL. The result supported the conclusion that the ACL cells are able to form more stable adhesion plaques than the MCL cells.

Proliferative differences between ACL and MCL cells have been well demonstrated. The outgrowth of cells from ACL explants was slower than that from MCL explants and slower in closing in vitro confluent culture streak wounds [45]. Growth curves of ACL and MCL cultures at both passage numbers two and six showed a significantly slower rate of proliferation of ACL cells than MCL cells (Fig. 23.8) [47–49]. DNA synthesis measured in terms of tritiated thymidine incorporation of both log phase and confluent cultures supports the conclusion that differential proliferation rates of these cells exist in culture [45]. Furthermore, an in vitro wound created in a confluent layer of ACL and MCL cells revealed that 48 h after injury, the cell-free zones created in ACL cultures were occupied partially by single cells in a nonconfluent fashion. In contrast, the wounded zone in the MCL cultures was almost completely covered by cells [45] (Fig. 23.9). These results demonstrate a lower proliferation and migration potential of ACL cells in comparison with MCL cells in response to injury.

Another factor important in the migration of ligament fibroblasts is the expression of fibronectin [50]. The ACL and PCL each contain twice the amount of fibronectin found in either MCL or patellar tendon. The cellular-biologic characteristics of phenotype, cellular alignment and crimp pattern, proliferation, and migration reveal differences in the intrinsic properties of the normal ACL versus MCL cells. These different intrinsic properties between the cells of these ligaments have been proposed as important factors in their differential repair mechanisms.

Ligamentous injuries of the knee joint are among the most common ligament injuries encountered by orthopedists [51]. The anterior cruciate ligament and medial collateral ligament are major ligaments contributing to the stability and normal functioning of the knee joint [52–55]. Injuries to these ligaments can be clinically classified as described by Rockwood et al. [56] into three degrees. A first degree sprain involves a tear of a minimum number of fibers (microtears) or less than one-third of the ligament. There is minimal hemorrhage and swelling, localized tenderness, and no clinical instability or laxity. Second degree sprains involve a tear of more ligamentous fibers (one-third to two-thirds of the ligament) with a greater loss of function, localized tenderness, and an effusion, but there is no laxity or noticeable instability. Third degree injuries have greater disruption (greater than two-thirds of the ligament), more tenderness, and demonstrable laxity of the knee joint. Laxity of the knee can be evaluated clinically on a grade 1–3 scale (grade 1 = 0–5 mm, grade 2 = 5–10 mm, grade 3 = >10 mm). Gradation is dependent on the distance of translation of the joint on physical exam, reflecting injury severity in the ligament.