Characterization of Immature Knee Joint Tissues Figure 1. Continuum of knee joint connective tissues. Based on their structural compositions, the major connective tissues of the knee joint can be conceptualized along a continuum from hyaline (condylar and patellar cartilage), to fibrocartilaginous (meniscus), to fibrous (ligament). doi:10.1371/journal.pone.0026178.g001 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 glycosaminogly- cans (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 cross- linking 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 applica- tions. 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. The objective of this study was to perform a comprehensive characterization of the tensile properties, collagen content, and pyridinoline crosslink abundance of the major connective tissues of the immature bovine knee joint. Tissues of interest were femoral condylar and patellar cartilage, medial and lateral menisci, cranial and caudal cruciate ligaments (analogous to the ACL and PCL in humans, respectively), medial and lateral collateral ligaments, and patellar ligament. It was hypothesized that trends in tensile properties would reflect those in collagen content; that tensile properties and collagen content would be higher in fibrocartilag- inous and ligamentous tissues than in hyaline tissues; and that pyridinoline crosslinks would be found in all tissues, in spite of the immaturity of the tissues. Results from this investigation reinforce the interplay of tissue biomechanics and biochemical content and provide design parameters for future efforts concerned with connective tissue engineering for joint repair. Materials and Methods Tissue harvest and specimen preparation Tissue specimens were harvested from the knee joints of 6 one- week-old male bovine calves (Research 87, Boston, MA), shortly after slaughter of the animals for commercial use in the food industry. To normalize variability among animals, each leg came from a different animal. Hyaline femoral condylar cartilage (CC), hyaline patellar cartilage (PC), medial meniscus (MM), lateral meniscus (LM), cranial cruciate ligament (CraCL), caudal cruciate ligament (CauCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), and patellar ligament (PL) were taken. For CC and PC specimens, the cartilage was separated from subchondral bone with a scalpel. For MM and LM specimens, the femoral and tibial surfaces, as well as the inner 1/3 and outer 1/3 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26178