The knee is a pivotal hinge joint that permits flexion, extension, and limited rotation through coordinated action of its hyaline, fibrocartilaginous, and fibrous connective tissues. Hyaline cartilage is found at the condylar surfaces of the femur and tibia, as well as on the patella. Fibrocartilage comprises the medial and lateral menisci, which are crescent-shaped structures interposed between the femoral and tibial condyles. Fibrous tissue makes up the major ligaments of the knee joint, in particular the patellar ligament, the collateral ligaments, and the cruciate ligaments. The patellar ligament provides stability to the patella as it glides over the patellofemoral groove and femoral condyles. The medial and lateral collateral ligaments (MCL and LCL) are extracapsular ligaments that protect the medial and lateral sides of the knee from a contralateral outside or inside bending force, respectively. The anterior and posterior cruciate ligaments (ACL and PCL) are intracapsular ligaments that stabilize the knee during rotation and bending. Together, these tissues contribute significantly to normal knee function.

The connective tissues of the knee joint are known to derive their mechanical properties from their biochemical components, but precise structure-function relationships remain elusive beyond general notions of the role of the extracellular matrix (ECM). Structurally, each of these tissues is hypocellular and possesses an ECM rich in collagen, with varying amounts of glycosaminoglycans (GAGs) [4], [5]. In general, collagen is known to be largely responsible for the tensile integrity of these tissues, while GAGs, predominant in hyaline cartilage and sparse in fibrous tissues, contribute to compressive strength [6]. In addition to total collagen content, the amount of crosslinking present in the collagen network has been shown to play an important role in tissue tensile properties [7]. In examining tissue tensile properties, two important measures of tensile integrity are Young's modulus and ultimate tensile strength (UTS). Young's modulus is a measure of a material's tensile stiffness, and the UTS is the maximum stress a material can withstand. Though collagen content and crosslinking are known to play a role in tensile mechanics, their precise structure-function relationships with respect to Young's modulus and UTS remain unclear. Pyridinoline crosslinks have been shown to correlate with both tensile strength and stiffness in articular cartilage [8], but there is a dearth of literature describing the contribution of pyridinoline crosslinks to the mechanical behavior of fibrocartilage or ligament tissues.

In humans, conditions afflicting the connective tissues of the knee, such as traumatic injury and osteoarthritis, contribute to substantial healthcare costs and work-related disability [9]–[11]. The field of tissue engineering aims to improve orthopaedic medicine by providing functional replacements for damaged or diseased joint tissues. Recent tissue engineering efforts have focused on major connective tissues such as hyaline cartilage [12], [13], meniscus [14], [15], tendon [16], [17], and ligament [18]. Although various approaches have been employed to engineer these tissues, it has been difficult to reproduce native collagen organization and attain native mechanical properties. Various types of mechanical [13], [19]–[21] and biochemical [22], [23] stimuli have been studied to improve construct properties, and both scaffold-free [12], [24], [25] and scaffold-based [26], [27] approaches have been investigated for connective tissue engineering applications. An additional consideration in these tissue engineering efforts has been the cell source used to produce constructs. Comparisons of cell types have shown that immature cells exhibit increased biosynthesis [28], making them promising candidates for tissue engineering. Immature cells have been used to produce constructs with clinically relevant dimensions [12] and mechanical properties on par with native tissue. To make informed cell source choices, it is necessary to establish a comprehensive understanding of the physiology of immature joint tissues. Moreover, while studies on the knee joint are well represented in the literature, it is important to note that much of what is known about the structure-function relationships of these tissues comes from assessments of adult rather than immature joints, whether human or animal. Given the prevalence of knee injuries in the pediatric population [29], along with a greater push towards using immature tissues as cell sources for tissue engineering, a thorough elucidation of the biochemistry of immature knee joint tissues, not just adult tissues, is warranted. An understanding of immature joint physiology may also yield insight into tissue development by providing a reference to which adult tissues can be compared, as well as informing a general understanding of factors at play in pediatric joint injury. Additionally, because orthopaedic explant and tissue engineering studies are relying more readily on bovine tissues [12], [20], [30]–[32], it is imperative that a full assessment of the bovine joint be undertaken.

In the present study, tissues of interest were first examined histologically for the presence of collagen and GAGs to infer qualitative structural differences underlying the biomechanical distinctions between these different tissues. Meniscus and ligament specimens appeared nearly identical, exhibiting extensive staining for collagen with no observable GAG staining (Figure 2). Hyaline cartilage, by contrast, exhibited less collagen staining than either meniscus or ligament, but also significant GAG staining. These histological trends correspond to the notion of knee joint connective tissues spanning a continuum between hyaline tissue (high collagen, high GAG) and fibrous tissue (high collagen, low GAG) (Figure 1). These qualitative histological differences relate to the functional roles of these tissues: fibrous tissues (ligaments and tendons) and fibrocartilage tissues (menisci) experience tremendous tensile stresses during locomotion, while hyaline cartilage experiences a balance of both tensile and compressive stresses, though preferentially the latter.

Tissue tensile properties, especially in connective tissues, are derived in part from collagen content [6], as well as from other matrix components, such as elastin [35]; therefore, it was hypothesized that trends in tensile properties would reflect trends in collagen content. In this study, collagen content was quantified in each tissue and normalized to tissue wet weight (Figure 3A). It was found that the menisci had the highest collagen content, followed by the patellar ligament and the collateral ligaments. Collagen content was lowest in the hyaline cartilages and the cruciate ligaments. As expected, the tensile properties (Figure 4) appear to reflect the general trends observed in collagen content normalized to wet weight. In particular, it was found that the menisci and patellar ligament exhibited significantly higher stiffness (Young's moduli) and strength (UTS) values compared to the other tissues, while the hyaline cartilages and the cruciate ligaments were among the softest and weakest in tensile properties.

The differences in tensile properties among the ligament tissues (high in patellar ligament, medium in collateral ligaments, and low in cruciate ligaments) may reflect the anatomical development of these tissues, since the stiffer/stronger tissues are extracapsular ligaments, and the softer/weaker tissues are intracapsular ligaments. In particular, the patellar ligament arises from fibers of the quadriceps muscle attaching inferiorly to the tibial tuberosity, hence the term “patellar tendon” often used interchangeably with patellar ligament, given the tendinous origin; the cruciate ligaments develop posteriorly from the articular interzone; and the collateral ligaments form independently of the joint capsule (LCL) or from mesenchymal condensation in the joint capsule (MCL) [36]. Furthermore, of particular interest was the finding that CraCL is significantly softer and weaker than CauCL. Future studies should seek to examine whether this relationship is maintained in adult cows, as well as whether it is observed in humans (i.e., between the ACL and PCL). Taken together, the tensile data described above contribute important information about the tensile properties of immature tissues, especially in light of the increasing incidence of knee joint injuries among youths [29]. Additionally, these tensile properties may serve as important benchmarks to determine success criteria for in vitro engineering of the major knee joint connective tissues, all of which play important roles in mechanical function. Tissue engineering efforts aimed at recapitulating native tissue structures should strive to reproduce native tissue biomechanical properties, as well.

Crosslink analysis with HPLC showed that the different joint tissues had varying pyridinoline abundances that contributed to tensile stiffness. The data showed that the hyaline cartilages and the cruciate ligaments exhibited the highest pyridinoline levels (Figure 3). Both the patellar ligament and CauCL exhibited higher tensile stiffness values that paralleled pyridinoline content but not the amount of collagen. Although pyridinoline has been shown to correlate with tensile strength and stiffness in bovine articular cartilage [8], this is the first study to show that pyridinoline also contributes to the mechanical properties of other joint tissues. These results also corroborate structure-function relationships in other species. For example, a study of the rat tendon demonstrated that pyridinoline was a better indicator of ultimate stress than collagen content [37]. These structure-function relationships illustrate the importance of crosslinking in a variety of joint tissues.

Pyridinoline content is known to generally increase as tissues matures, but this study provides comprehensive, quantitative benchmarks that can be compared to adult tissue values. For instance, the observed pyridinoline abundances for condylar cartilage and meniscus fibrocartilage are approximately 50% and 70% of the mature values, respectively [8], [38]. These pyridinoline results can inform future tissue engineering efforts that aim to reproduce the biochemical composition of native tissues. Because engineered cartilage has shown less collagen crosslinking than native tissue, strategies such as increasing lysyl oxidase expression [39] may be needed to increase pyridinoline formation. Other stimuli such as TGF-β1 have been shown to increase pyridinoline content in articular cartilage [40] and could potentially be beneficial for enhancing crosslinking in engineered tissue as well. Considering the role of pyridinoline in tissue mechanics [8], [41] and the inherently mechanical nature of knee joint connective tissues, crosslinking should be a central focus of future tissue engineering approaches.