portions of the annulus, were sliced away, leaving the approximate interior circumferential portion of the specimen for assessment. CraCL, CauCL, MCL, LCL, and PL were taken whole from their attachments. From each freshly harvested specimen, a 3 mm dermal biopsy punch was used to obtain samples for histology, quantitative biochemistry, and high performance liquid chromatography (HPLC). The remainder of each specimen was then prepared for tensile testing. Tensile specimens were stored for a maximum of 24 h in phosphate buffered saline with protease inhibitors at 4uC and were allowed to equilibrate to room temperature prior to testing. Histology Samples were cryo-embedded and sectioned at 14 mm. Sections were fixed in formalin for 10 min and then stained with either picrosirius red or safranin O/fast green as described previously [12]. Samples were dehydrated in an ascending series of ethanol and mounted with coverslips prior to imaging. Quantitative biochemistry Biochemistry samples were weighed wet, frozen, lyophilized for 48 h, and then digested in a phosphate buffer with 125 mg/mL papain (Sigma) for 18 h at 65uC. A chloramine-T hydroxyproline assay was employed to quantify total collagen content after 2 N NaOH hydrolysis for 20 min at 110uC [33]. Total collagen was normalized to tissue wet weight and tissue dry weight. High performance liquid chromatography (HPLC) HPLC was performed to quantify the abundance of pyridinoline crosslinks. Samples were weighed wet, digested in 800 mL of 6 N HCl at 100uC for 20 h, and then dried using a vacuum concentrator. Samples were re-suspended in 50 mL of an aqueous solution containing 10 nmol pyridoxine/mL and 2.4 mmol homoarginine/ mL and then diluted fivefold with an aqueous solution of 0.5% HFBA acetonitrile in 10% acetonitrile. 10 mL of each sample was injected into a 25 mm C18 column (Shimadzu, Columbia, MD) and eluted using a solvent profile described previously [34]. To quantify the amount of crosslink in each sample, pyridinoline standards (Quidel, San Diego, CA) were employed to create a calibration curve. Characterization of Immature Knee Joint Tissues comparison to an in vivo context, it was important to maintain consistent mechanical testing procedures across tissue types so that comparisons could be made between tissues without the risk of introducing confounding variables. The specimen was photo- graphed alongside a ruler, and ImageJ software was used to determine the width and thickness. A uniaxial electromechanical materials testing system (Instron Model 5565, Canton, MA) was employed to determine tensile properties with a 50 N (CC and PC only) or 5 kN load cell (all other tissues). CC and PC specimens were affixed with cyanoacrylate glue to paper tabs outside of the gauge length for gripping; all other specimens were gripped directly outside of the gauge length. MM and LM specimens were tested in the circumferential direction. CraCL, CauCL, MCL, LCL, and PL specimens were tested in the longitudinal direction. Tensile tests were performed until failure within the gauge length at a strain rate of 1% of the gauge length per second. Force- displacement curves were generated, and stress-strain curves were calculated by normalizing data to specimen dimension. The apparent Young’s modulus, a measure of specimen tensile stiffness, was determined by least squares fitting of the linear region of the stress-strain curve. The ultimate tensile strength (UTS) was determined as the maximum stress reached during a test. Statistical analysis All biochemical, HPLC, and tensile assessments were made using n = 5–6. To compare among tissues, a single-factor analysis of variance was employed, and a Fisher least significant difference post hoc test was used when warranted. Significance was defined as p,0.05. Results Histology Representative histology for hyaline cartilage, meniscus, and ligament are shown in Figure 2. Staining for collagen was observed in all tissues, though hyaline cartilage exhibited less extensive collagen staining compared to either meniscus or ligament. Extensive staining for GAG was observed in the hyaline cartilage specimens, but was not qualitatively observed in meniscus or ligament specimens. Tensile testing Each specimen was cut into a dog-bone shape with a 1-mm-long gauge length. Although this tissue preparation may limit Collagen content The collagen/wet weight for CC, PC, MM, LM, CraCL, CauCL, MCL, LCL, and PL were 6.762.6%, 5.161.4%, Figure 2. Histology of representative joint tissues. Picrosirius red staining for collagen showed that hyaline cartilage, meniscus, and ligament all had significant collagen content. The meniscus and ligament samples stained more intensely for collagen than hyaline cartilage. Safranin O/fast green staining for GAG showed that hyaline cartilage had significant GAG content; meniscus and ligament did not exhibit GAG staining. doi:10.1371/journal.pone.0026178.g002 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26178