Periodic Reporting for period 1 - DYNAMIN (Deciphering collagen mineralization process by dynamic imaging in liquid)
Periodo di rendicontazione: 2021-04-01 al 2023-03-31
This MSC Action entitled ‘Deciphering collagen mineralization process by dynamic imaging in liquid’, aimed to gather dynamic information about the process of bone mineralization at the nanometer scale.
The mechanisms governing bone formation, degradation and regeneration are still poorly understood, despite its clinical relevance. The social impact of a good bone health is remarkable, being a fundamental factor in ensuring a healthy aging population. The yearly economic burden of osteoporotic fractures in the EU is estimated at € 37 billion and expected to increase to € 47.4 billion in 2030. This fact indicates an imperative need to understand the mechanisms that determine bone mineralization and degradation, and to translate this knowledge to the clinics.
The project was divided in two main parts: to unravel the dynamics of the mineral deposition pattern through the extracellular matrix; and to prove the role of one of the main proteoglycans (biglycan) as a promoter of mineralization.
The mechanisms governing bone formation, degradation and regeneration are still poorly understood, despite its clinical relevance. The social impact of a good bone health is remarkable, being a fundamental factor in ensuring a healthy aging population. The yearly economic burden of osteoporotic fractures in the EU is estimated at € 37 billion and expected to increase to € 47.4 billion in 2030. This fact indicates an imperative need to understand the mechanisms that determine bone mineralization and degradation, and to translate this knowledge to the clinics.
The project was divided in two main parts: to unravel the dynamics of the mineral deposition pattern through the extracellular matrix; and to prove the role of one of the main proteoglycans (biglycan) as a promoter of mineralization.
During the first year of the project, optimization of the sample preparation protocols and the liquid phase imaging protocols were carried out. Different demineralization and high-pressure freezing vitrification protocols were applied to maximize vitrification depth and sample quality. Cryo-sections thickness, thawing, and remineralization protocols were optimized until the entirely workflow proceeded smoothly. Mineralization experiments were initiated in the LPEM cell by introducing an artificial osteogenic medium at a low and constant flow.
The detection of the early mineral deposits was mainly carried out in STEM mode. However, the interactions of the electrons through the water column (inelastic scattering) lead to the broadening of the electron probe, producing a blurry effect in the image and reducing the spatial resolution. In STEM, beam broadening increases with the depth of the focal plane, limiting the position of the objects that can be imaged. The visualization of mineral particles was achieved, although some optimization of the imaging parameters is still required.
The detection of the early mineral deposits was mainly carried out in STEM mode. However, the interactions of the electrons through the water column (inelastic scattering) lead to the broadening of the electron probe, producing a blurry effect in the image and reducing the spatial resolution. In STEM, beam broadening increases with the depth of the focal plane, limiting the position of the objects that can be imaged. The visualization of mineral particles was achieved, although some optimization of the imaging parameters is still required.
Although complete visualization of the mineralization process has not yet been achieved, considerable progress has been made in this first year of research. In parallel, a series of experiments were initiated in which the liquid sample was encapsulated with a graphene layer, instead of using the LPEM holder. This technique has reported a considerable increase in spatial resolution, being possible to visualize early mineral foci in hydrated conditions with nanometer resolution.