This study aimed to characterize the in-situ mechanical property and morphology of individual collagen fibril in osteoarthritic (OA) cartilage using indentation-type atomic force microscopy (IT-AFM). The specimens with intact articular cartilage (AC), mild to severe degenerated OA cartilage were collected with informed consent from the postmenopausal women who underwent hip or knee arthroplasty. The fresh specimens were cryo-sectioned by layers with 50m thick for each from the articular surface to calcified cartilage, and then processed for AFM imaging and nanoindentation test. For each layer, a total of twenty collagen fibrils were randomly selected for testing. AFM tips with the nominal radius less than 10 nm were employed for probing the individual collagen fibril, and the obtained cantilever deflection signal and displacement were recorded for calculating its elastic modulus. Besides AFM nanoindentation, AFM and scanning electron microscopy (SEM) images, haematoxylin & eosin (H&E) staining and micro-indentation were performed on AC to study the changes of ultrastructure and composition between intact AC and OA cartilage. Results showed that an intact AC exhibited a gradation in elastic modulus of collagen fibrils from surface region (2.65±0.31GPa) to bottom region (3.70±0.44GPa). It was noted in the initial stage of OA cartilage that the coefficient of variation for mechanical properties of collagen fibers, ranging from 25~48%, significantly increased as compared with intact one (12%). The thickened and stiffened collagen fibrils initially occurred at either surface region (3.11±0.91GPa) or bottom region (5.64±1.10GPa) with OA progression. Besides thickens, alteration of D-periodic banding patterns of collagen fibrils was observed. It was echoed by fibrotic changes of surface region and tidemark irregularities. On the contrast, the micromechanical properties of cartilage decreased while AC suffered from OA. This result revealed the different approachs of nano and micro-mechanical properties changes in AC. In summary, the alteration of mechanical properties of collagen fibrils started from calcified cartilage as well as articular surface during OA onset, and the low compliance of thickened collagen fibrils deteriorated along disease progression. This study also reveals that the outstanding ability by AFM, in investigating the structure and mechanical properties of collagen fibrils and AC in nanometer scale, is impressive and this nanotechnological instrument is worth to be expected in further development for clinical use. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

Tissue engineering holds great promise for developing functional tissue to repair damaged articular cartilage. However, cartilaginous tissues formed in vitro typically possess poor mechanical properties in comparison to native tissue due to the inability of chondrocytes to accumulate adequate amounts of extracellular matrix (ECM). While mechanical stimuli can enhance the properties of engineered cartilage, it may be more efficient to harness the underlying mechanotransduction pathways responsible. Substantial evidence suggests that the mechanotransduction signaling cascade is initiated by rapid adenosine 5’-triphosphate (ATP) release from chondrocytes (purinergic receptor pathway). Thus, the purpose of this study was to investigate the effects of exogenous ATP supplementation on the biochemical and mechanical properties of tissue engineered cartilage. Primary bovine articular chondrocytes, seeded on MilliporeTM filters, were grown in the presence of 0, 62.5 or 250 µM ATP for a period of four weeks. Both anabolic and catabolic effects were examined and a therapeutic dose range of ATP was determined. ATP stimulation (62.5 - 250 µM) enhanced ECM synthesis by 23 - 43% and long-term collagen accumulation by 16 - 26%, in a dose-dependent manner; however, long-term proteoglycan accumulation decreased as a result of 250 µM ATP. In addition, ATP supplementation significantly improved the mechanical properties of the developed tissues (5- to 6.5-fold increase in tissue stiffness). Interestingly, high doses of ATP (250 µM) also elicited a 2-fold increase in MMP-13 gene expression, 39% increase in MMP-13 activity, and 54% more extracellular inorganic pyrophosphate (ePPi) – an ATP degradation product. Dose-dependent increases in MMP-13 activity suggested that catabolic effects were occurring alongside anabolic effects, which initiated the investigation of a therapeutic dose of ATP. Doses of 31.25 µM and 125 µM ATP were added to cartilaginous tissues and investigated in terms of MMP-13 activity and ECM synthesis. Tissues supplemented with 62.5 – 125 µM ATP exhibited a balance between anabolic and catabolic effects. By harnessing the purinergic receptor pathway, anabolic effects of mechanical stimuli were achieved in the absence of externally applied forces. Understanding how the catabolic effects of ATP are manifested would be valuable in order to further maximize the therapeutic potential of ATP stimulation. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2010-09-22 14:08:28.114

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Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on June 24, 2009. Major professor: Dr. Scott Hazelwood. "Presented to the faculty of the College of Engineering, California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in General Engineering." "June 2009." Includes bibliographical references (p. 72-76). Also available on microfiche.

Thesis (M.S.)--California Polytechnic State University, 2008. / Mode of access: Internet. Title from PDF title page; viewed on January 29, 2009. Major professor: Stephen Klisch, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Mechanical Engineering." "December 2008." Includes bibliographical references (p. 56-58). Also available on microfiche.

Thesis (Ph.D.)--Drexel University, 2010. / Includes abstract and vita. Includes bibliographical references (leaves 188-218).

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains x, 114 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 69-77).

Studies have investigated and dismissed the effect of freeze-thaw cycles on both skeletal muscle and on trabecular bone, but have failed to properly address the effects of these storage methods on the integrity of articular cartilage. Preventing cartilage injury is important in minimizing the long term debilitating effects of osteoarthritis. Accurate subfracture injury prediction must take into account the possible effects that freeze thaw cycles may have on the mechanical properties of cartilage tissue. This paper addresses this concern with matched pair testing of various low temperature storage techniques against fresh control groups. Controlled mechanical indention tests were performed on bovine articular cartilage-on-bone specimens to compare stiffness, peak stress, and loading energy of the cartilage. Findings showed that a slow freeze thaw or flash freeze cycle caused cartilage stiffness to decrease by 37% and 31% respectively. Compressive stress at this strain was also lowered by 31% with a single freezing process. These results may be indicative of a weakened extracellular matrix structure caused by the freeze-thaw process. It is still unclear whether these changes in mechanical properties will result in a change in injury susceptibility for articular cartilage. / Master of Science