Morphological and chemical changes in in vitro bone mineral and the effect of strontium on in vitro mineralisation
Creators: name: Nitiputri, Kharissa Ayu Perdana
Publisher: name: Imperial College London
Year: 2015

description: The steps involved in early bone mineralisation have been extensively studied. Studies have suggested various means by which bone mineralisation occurs: (1) a cell-independent (protein-assisted) process where non-collagenous proteins mediate soluble ions to form mineral on the collagen fibril; (2) a cell-controlled mechanism where mineral seeds formed within intracellular vesicles released from the plasma membrane would subsequently rupture and disperse their contents on the extracellular matrix; and (3) an acellular route in which amorphous calcium phosphate mineral precursors are produced and deposited in collagen fibrils where they transform into more crystalline apatite platelets. Despite extensive studies, there are still unanswered questions about how bone becomes bone. The first series of experiments in this thesis are aimed at studying the mineral characteristics of early in vitro osteoblast mineralisation at the extracellular matrix. These experiments seek to determine the sequence of possible mineralisation events that take place during mineral nucleation and growth on collagen. Transmission electron microscopy was used to provide high spatial resolution, which was compounded with chemical analysis of energy dispersive x-ray and electron energy-loss spectroscopies. We identified carbonate-rich calcium phosphate dense granules in the extracellular matrix that may act as seeds for growth into larger, submicron-sized, globular aggregates of apatite mineral with a different stoichiometry. These globules appear to mineralise the collagen fibrils forming crystalline textured crystals with higher calcium-to-phosphate ratio and lower carbonate content as the mineral phase of bone. We provide new evidence that the use of a carbonate rich, amorphous calcium phosphate spherical bioseed could be a process by which a soluble calcium phosphate phase is stabilised and delivered to the collagen for subsequent maturation and collagen mineralisation. We also examined the effect of strontium ion supplementation to bone mineralisation as a translational study. Previous studies showed the positive effects of in vivo strontium supplementation as an anti-osteoporotic drug. Strontium is able to: (1) stimulate bone formation; (2) increase osteoid surface, osteoblast surface, and bone forming surfaces; (3) decrease bone resorbing cells; (4) increase bone strength and mass; and (5) reduce the risk of fractures. In the in vitro system, studies have focused on how strontium ions increase bone mineral’s a- and c-axis lattice parameters. The effect of strontium ions on the matrix component of bone is the aim for the second part of this thesis. Using Raman spectroscopy, TEM imaging, and biochemical quantification, we studied the effect of strontium ion supplementation on in vitro MC3T3 osteoblasts, with close focus on the osteoblast matrix. We observed that cultures treated with high strontium supplementation had impaired mineralisation, where nodules were formed but failed to mineralise. Periodic collagen banding was seen on TEM micrograph from all treatments. Collagen organisation was quantified using image analysis of TEM micrograph, and strontium supplementation seemed to affect fibril organisation. A slight addition of 0.1 mM strontium seemed to result in the less random organisation of collagen fibril, while 3 mM supplementation seemed to increase the random organisation. Only cultures treated with the highest amount of strontium supplementation showed an abundance of matrix vesicles around the collagen fibrils. Raman spectroscopy showed an increase in lipid detection on strontium supplemented groups, which may be due to the increased presence of matrix vesicles. Taken together, high strontium supplementation may decrease the rate of degradation of matrix vesicles and lead to altered mineralisation whereby nodules form but fail to mineralise. The relative amounts of collagen were also explored by Raman spectroscopy and hydroxyproline analysis; however, our findings were not statistically significant. Further experiments are needed to more completely elucidate the molecular mechanisms at play in strontium’s effect on bone mineralisation.