The origin of lamellar structure in cortical bone

Bone maturation is a highly complex process in vivo where several cellular events are still not completely understood. An example of this is the production of new unmineralized bone, or osteoid, by osteoblasts in cortical bone. The osteoid is subsequently mineralized into new bone in a step-wise process, which results in a structural appearance of layers of bone with different thickness. The theory investigated in this project is that the osteoblasts are guided in their osteoid production by the degree of mineralization of the secreted osteoid and that they preferably produce osteoid on a stiffer mineralized surface. To evaluate the influence of substrate stiffness on osteoblast development and osteoid production, pre-osteoblast MC3T3-E1 cells were cultured onto collagen type I - coated polyacrylamide (PA) substrates with stiffness 6.4 ± 0.7 kPa (soft), 72.9 ± 1.7 kPa (medium) and 135.9 ± 3.4 kPa (stiff). Type I collagen coated tissue culture polystyrene, with and without addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), were applied as positive controls. Total cellular content of RNA and DNA for the MC3T3-E1 cells cultured onto the different substrates were determined, but no significant impact of substrate stiffness on the amount of total RNA and DNA could be detected after 48 hours of culture. The amount of total protein content for the MC3T3-E1 cells on the different substrates were too low to be detected. However, staining for alkaline phosphatase revealed a higher degree of differentiation among the MC3T3-E1 cells seeded onto the stiff substrate and plastic controls already after 24 hours. An up-regulation in gene expression of collagen type I, bone sialoprotein and osteopontin was further observed for the collagen coated plastic controls but no difference could be detected between the three sample substrates after 48 hours. Osteoblasts experience a range of partly mineralized surfaces during the bone formation process in vivo and to mimic this pieces of cow bone were demineralized with EDTA for 6, 12, 24, 48 and 72 hours and further for 4, 7, 11, 14, 18 and 21 days. These were applied as alternative MC3T3-E1 cell substrates together with controls lacking demineralization. Staining to evaluate impacts of the demineralization treatment on seeded MC3T3-E1 cell after 48 hours revealed an increased degree of demineralization and presence of un-mineralized flakes associated with prolonged EDTA treatment. Significant higher levels of total RNA and DNA were detected for the control bone pieces lacking demineralization compared to the bone pieces with demineralization but the protein levels were also in this case below the limit of detection. In general, stiffer substrates were shown to induce higher level of differentiation and proliferation among osteoblasts but no impact of substrate stiffness on metabolic activity could be identified. The substrate stiffness was shown to affect osteoblast maturation, with an accelerated process observed for the stiffer substrates, but more research is needed before any conclusions can be drawn considering the impact of substrate stiffness on osteoblasts during the formation of lamellar structure in cortical bone.