Tensile Properties, Collagen Content, and Crosslinks in Connective Tissues of the Immature Knee Joint Sriram V. Eleswarapu1,2., Donald J. Responte1., Kyriacos A. Athanasiou 3 * 1 Department of Bioengineering, Rice University, Houston, Texas, United States of America, 2 Medical Scientist Training Program, Baylor College of Medicine, Houston, Texas, United States of America, 3 Department of Biomedical Engineering, University of California, Davis, California, United States of America Abstract Background: The major connective tissues of the knee joint act in concert during locomotion to provide joint stability, smooth articulation, shock absorption, and distribution of mechanical stresses. These functions are largely conferred by the intrinsic material properties of the tissues, which are in turn determined by biochemical composition. A thorough understanding of the structure-function relationships of the connective tissues of the knee joint is needed to provide design parameters for efforts in tissue engineering. Methodology/Principal Findings: The objective of this study was to perform a comprehensive characterization of the tensile properties, collagen content, and pyridinoline crosslink abundance of condylar cartilage, patellar cartilage, medial and lateral menisci, cranial and caudal cruciate ligaments (analogous to anterior and posterior cruciate ligaments in humans, respectively), medial and lateral collateral ligaments, and patellar ligament from immature bovine calves. Tensile stiffness and strength were greatest in the menisci and patellar ligament, and lowest in the hyaline cartilages and cruciate ligaments; these tensile results reflected trends in collagen content. Pyridinoline crosslinks were found in every tissue despite the relative immaturity of the joints, and significant differences were observed among tissues. Notably, for the cruciate ligaments and patellar ligament, crosslink density appeared more important in determining tensile stiffness than collagen content. Conclusions/Significance: To our knowledge, this study is the first to examine tensile properties, collagen content, and pyridinoline crosslink abundance in a direct head-to-head comparison among all of the major connective tissues of the knee. This is also the first study to report results for pyridinoline crosslink density that suggest its preferential role over collagen in determining tensile stiffness for certain tissues. Citation: Eleswarapu SV, Responte DJ, Athanasiou KA (2011) Tensile Properties, Collagen Content, and Crosslinks in Connective Tissues of the Immature Knee Joint. PLoS ONE 6(10): e26178. doi:10.1371/journal.pone.0026178 Editor: Alejandro Almarza, University of Pittsburgh, United States of America Received August 1, 2011; Accepted September 21, 2011; Published October 13, 2011 Copyright: 2011 Eleswarapu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was performed with funding from grants R01 AR053286 and R01 AR047839 by the National Institutes of Health, as well as UC Davis institutional funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: athanasiou@ucdavis.edu . These authors contributed equally to this work. Introduction The major connective tissues of the knee joint act in concert during locomotion to provide joint stability, smooth articulation, shock absorption, and distribution of mechanical stresses [1-3]. These functions are largely conferred by the intrinsic material properties of the tissues, which are in turn determined by their biochemical compositions. Based on structure-function relation- ships, each connective tissue of the knee joint can be conceptu- alized along a continuum from hyaline to fibrocartilaginous to fibrous (Figure 1). These tissues have received considerable attention in both basic science and clinical literature, but much work remains to be done to elucidate the contributions of particular biochemical components to important mechanical parameters, especially with respect to applications in tissue engineering. Approaches in tissue engineering are guided heavily by the interplay of native tissue structures and their corresponding functional correlates. To better understand these relationships, this study examines the biochemical composition and tensile properties of the major connective tissues of the immature bovine knee joint. 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 PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26178