Anterior cruciate ligament (ACL) injuries are one of most common musculoskeletal injuries and negatively affect mobility and quality of life. ACL rupture requires reconstruction to repair ligament at an estimated cost of $1.5 billion/year. Current surgical solutions invariably involve either donor site morbidity with the use of autografts or the risk of disease transmission and immune rejection with the use of allografts. Successful reconstruction requires the presence of an intact interface between ligament and bone, a transitional tissue called the enthesis. The enthesis is critical for the safe and effective transfer of force from the stiff bone to the more compliant ligament by providing a gradual transition of mechanical and biochemical properties to prevent the formation of stress concentrations. A tissue engineered ligament containing mature entheses is a promising alternative to autografts and allografts, especially since this interface does not normally regenerate. Toward this end, this dissertation sought to improve engineered fibrin-based bone-to-bone ligaments previously developed by our lab and to demonstrate their utility in understanding physiological processes through three specific aims: 1) optimize the environment for in vitro ligament function, 2) induce the formation of a fibrocartilaginous interface, and 3) demonstrate the utility of engineered ligaments as a physiological model.
In Aim 1, the in vitro culture environment was investigated for engineered ligaments formed using human ACL fibroblasts. Using a DOE approach, we identified significant effects and interactions of soluble factors on the maximal tensile load (MTL) and collagen content of engineered human ACL. The DOE model was used to predict a maximal growth media which significantly improved the MTL and collagen content of engineered ligaments and can be combined with increases in the initial construct volume for 77% further improvement in MTL. In addition to the improvements in tissue function, these data suggest that a DOE approach can more efficiently optimize in vitro parameters including the dosage and timing of chemical and mechanical stimuli as well as any interactions.
Aim 2 presented two strategies to improve of the engineered enthesis. First, the local release of bone morphogenetic protein (BMP)-4 at the enthesis of engineered ligaments demonstrated improved interface strength as well as the transition of cells at the enthesis towards an unmineralized fibrocartilage phenotype. Second, engineered ligaments formed in a modular fashion improved the mechanical function and the morphology of the engineered enthesis including the development of cell and soft tissue integration into the mineral phase, a tidemark between mineralized and unmineralized tissue, and the presence of a dense band of extracellular matrix (ECM) at the soft tissue-mineral interface. Importantly, this is the first demonstration of the in vitro formation of a functional interface between engineered ligament and mineral in a complete bone-to-bone ligament unit.
Aim 3 demonstrated the use of our engineered ligament model as a physiological tool. During the estrogen surge in the menstrual cycle, there is an associated increase in the incidence of ACL ruptures as well as knee laxity. Using physiological levels of estrogen mimicking the estrogen surge in vitro, we determined that estrogen decreases the activity of the collagen crosslinker lysyl oxidase (LOX) with a subsequent decrease in tissue stiffness providing insight into why women have greater incidences of ACL rupture. We also examined the role of the exercise-induced biochemical environment on connective tissue using our in vitro model. Engineered ligaments cultured with serum obtained from human donors after exercise had significantly better mechanical strength and collagen content than those treated with serum obtained at rest. In 2D culture, we determined that this effect was likely a result of greater mTOR and ERK signaling.
In summary, the work in this dissertation has made great strides in developing a more mature engineered bone-to-bone ligament. We have optimized a growth factor environment for their in vitro culture and created the most advanced engineered enthesis to date. We have also used these engineered tissues as a platform to mechanistically study the influence of hormones on connective tissue. With further advances in our understanding of the in vivo development of ligaments and their entheses, our bone-to-bone engineered ligaments can be improved making them more suited for clinical applications and for probing physiologically processes in a more controlled environment.
|Commitee:||Reddi, A. Hari, Yamada, Soichiro|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||DAI-B 77/08(E), Dissertation Abstracts International|
|Keywords:||Anterior cruciate ligament, Enthesis, Fibrin, Ligament, Tissue engineering|
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