Ex Vivo Bone Culture: A Novel Method For Investigating Mechanical Loading Response And Osteocytes In Situ In Trabecular Bone

Osteocytes plays an important role in controlling and determining the function of the other cell types in bone, especially in response to mechanical loading and biochemical changes. The available data are still incomplete and much information derives from models that do not emulate the three dimensional structure of living bone. The use of the second generation Zetos™ system that enables long-term culture of trabecular bone ex vivo, together with the more traditional in vitro and in vivo models, will enable us to answer some of these knowledge gaps. A better understanding of osteocyte function specifically, and bone response to mechanical loading in general, can be obtained. In the first study, the second generation Zetos™ system was optimised and its software betatested in a series of trial experiments. The methodology and workflow used to prepare twenty four (24) ex vivo trabecular bone cores from a single bovine sternum, the maximum for each experiment, was described in great detail. Half of these bone cores had their marrow removed in the described experiment. Three (3) groups of four (4) bone cores, both with and without marrow, were loaded dynamically, with a load of 2,000 μstrain at 1 Hz, either for 100 cycles three times a day (100x3) or 300 cycles once a day (300x1) or not loaded (UL) respectively for 10 consecutive days. The bone cores without marrow showed significantly better increase in stiffness and response of their μCT histomorphometric parameters to loading over time, while their media have a consistently higher pH and lower ionic calcium (Ca²⁺) levels than that of the bone cores with marrow. Taken together, they indicated that removal of marrow in the bone cores was beneficial to the study of osteocytes in their native bone matrix environment using this Zetos™ system. This was possibly due to the improved fluid flow within the culture chambers when bone marrow was removed. In the second study, the use of the second generation Zetos™ system to investigate the application of a nano-engineered implant for local drug delivery in trabecular bone was described. The results demonstrated a consistent gradual release of a model drug, with a characteristic 3-dimensional distribution pattern, from the implant into the surrounding bone, over a 5-day period. The parameters that significantly affect the drug distribution were the flow rate of the bone culture medium, trabecular bone microarchitecture and mechanical loading of the bone core samples. This study demonstrated the usefulness of this system for drug release studies in ex vivo bone, which can be used to assist in the design of new drug delivery systems, and the optimisation of specific therapy delivery, in bone. The third study described the effect of exogenous human recombinant sclerostin (rhSCL) on the osteocytes of trabecular bone that were mechanically loaded ex vivo using this system. Bovine trabecular bone cores without marrow were subjected to daily episodes of dynamic loading and compared to unloaded bone cores or with the addition of exogenous rhSCL. Loaded bone showed an increase in apparent stiffness, calcein uptake (as a surrogate for Ca²⁺ influx), the Ca²⁺ and COOH-terminal telopeptide of type I collagen (β-CTX) levels in the perfusate, the mean osteocyte lacunar size (indicative of osteocytic osteolysis), and the expression of catabolic genes. These results indicated the direct contribution of osteocytes to bone mineral accretion and mechanical properties of trabecular bone, and support the concept that sclerostin acts directly on osteocytes to inhibit these effects via modulation of the osteocytic osteolysis process. In conclusion, the work done in this thesis was able to optimise the use of the second generation Zetos™ system for relevant bone and osteocyte-related research. This includes the investigation of drug delivery implants in large animal trabecular bone and as an additional tool for studying the osteocyte within its native lacuno-canalicular network. Further work using this system can assist in filling up important knowledge gaps on osteocytes mechanobiology and its role in the various metabolic functions of bone.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201