its preferential role over collagen in determining stiffness for certain tissues. 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 tremen- dous 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. Characterization of Immature Knee Joint Tissues 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-b1 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. This study provides biochemical and biomechanical data describing hyaline, fibrocartilaginous, and fibrous tissues of the immature bovine knee joint. These data elucidate important structure-function relationships that can inform directed approaches for functional connective tissue engineer- ing. In particular, future tissue engineering approaches should aim to incorporate methods for improving crosslinking, since crosslink abundance may be a more relevant predictor of tensile stiffness than collagen content for certain tissues, as evidenced by the relationships identified in the cruciate ligaments and patellar ligament. Future work may expand on this study by examining temporal development and maturation of the collagen network and tensile properties, or by making direct comparisons in pyridinoline crosslink abundance between immature and adult tissues. Finally, an assessment of these parameters in disease states such as osteoarthritis or traumatic injury models such as ligament rupture may shed light on predisposing factors. Author Contributions Conceived and designed the experiments: KAA SVE DJR. Performed the experiments: SVE DJR. Analyzed the data: KAA SVE DJR. Contributed reagents/materials/analysis tools: KAA. Wrote the paper: KAA SVE DJR. PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26178