Rapid and durable integration of articular cartilage via photochemical bonding : benchtop optimization, in-vitro analysis and preclinical model

Osteoarthritis (OA) is a degenerative disease of synovial joints in humans that affects the knee, hip, shoulder and wrists and is prevalent in over 30 million Americans over the age of 18 years. In the knee, OA affects the entire joint and is associated with the degeneration of articular cartilage and the menisci, subchondral bone sclerosis and inflammation of the synovial lining. OA is the most common form of arthritis and accounts for over $16.9 billion in hospitalization bills in the United States. OA is precipitated by several conditions including wear and tear, obesity and traumatic injuries caused to the joint. Untreated lesions or focal defects in articular cartilage are precursors to OA. Shortcomings in cartilage repair treatments motivate ongoing efforts to improve these strategies to enhance the implant-host interface. A common focal defect treatment is cartilage osteochondral autograft/allograft transplantation; however, inadequate graft integration with the surrounding native cartilage is a persistent challenge. This lack of integration can lead to poor mechanical load transfer, poor nutrient transport, fibrocartilage formation, tissue necrosis and continued cartilage degeneration. Photochemical tissue bonding is a promising strategy for improved graft integration that uses high wavelength light-activated photosensitizers to excite and crosslink tissue across the tissue interface via non-native bonds. The work described in this dissertation builds on initial proof of principle photochemical bonding techniques to integrate articular cartilage by delineating procedures to optimize the treatment protocol and bring its use closer to clinical implementation. The first and second parts of this thesis probe the different parameters that affect bonding shear strengths in articular cartilage. Four different photosensitizers with maximum light absorbance and activation at a high red wavelength, Methylene Blue (MB), Al(III) Phthalocyanine Chloride Tetrasulfonic Acid (CASPc), Aluminum Phthalocyanine Chloride (AlPc) and Verteporfin, were examined at different concentrations. Based on the bonding shear strengths achieved by the different photosensitizers over a range of concentrations, the exposure was examined for AlPc and verteporfin by either holding the light irradiance constant while varying the irradiation time or by holding the irradiation time constant and varying the irradiance. Results from these optimization experiments were used to further investigate bond durability and cell viability in environments similar to those found in a knee joint that has undergone a traumatic injury or surgical trauma. These environments include pro-inflammatory and anti-apoptotic environments with the addition of caspase inhibitors, which have been proposed to address the effects that injury or surgical trauma have on the joint. Results from these in-vitro experiments as well as those from the benchtop optimization were used to inform parameters used in a proof of principle preclinical in-vivo rabbit model. The benchtop optimization experiments showed that both AlPc and verteporfin produced substantial bonding with high shear strengths. Verteporfin, moreover, was able to produce higher bonding shear strengths at lower exposures than AlPc. Both AlPc and Verteporfin showed an increase in mean shear strength from intermittent concentrations with a decrease in bond strength at higher and lower concentrations. Light exposure studies showed that shear strength for both AlPc and Verteporfin was dictated by the total amount of exposure delivered to the interface instead of the rate of delivery or irradiance level. The in-vitro studies probed bond robustness, durability and cell viability in an artificially challenged environment. The use of interleukin-1, a pro-inflammatory signaling cytokine, was used at mild and moderate concentrations to test the robustness of the photochemical bonds. These experiments elucidated that photochemical bonds were not affected by this artificially challenged environment as exposure to IL-1 did not significantly alter the shear strengths. Lastly, the use of caspase inhibitors, such as ZVAD-fmk, have been proposed to be used in conjunction with cartilage repair techniques to form an apoptotic environment. Bond robustness was revealed to not be effected by such these inhibitors positively or negatively as no significance was shown between bond shear strengths between groups exposed or not exposed. Cell viability in all these in-vitro studies showed expected cell death near cut surfaces and live throughout with no clear differences between bonding and control groups. The preclinical study assessed photochemical bonding to enhance cartilage integration in an osteochondral allograft in an in-vivo rabbit model. At the two examined timepoints, the use of photochemical bonding did not cause any apparent macroscopic deterioration of the cartilage, menisci or surrounding tissues. This model showed osteochondral allografts stability was maintained over the period of 4 and 8-weeks. Osteochondral graft integration was evaluated nondestructively using optical coherence tomography to visualize osteochondral graph integration as well as via standard histological methods. No clear differences were noted amongst groups through these visualization techniques. The use of photochemical bonding to enhance cartilage integration is a viable and feasible option and supports further exploration of this strategy to augment clinical cartilage repair.

Keywords: Mechanical Engineering.
Advisor: Levenston Marc Elliot 
Chaudhuri Ovijit 
Yang Fan