Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep

Abstract

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 mm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (r parameter), and predominant orientation (c parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 mm. 2D maps of T and r overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus. ß

Porous scaffold architecture guides tissue formation

“…This suggests that a higher degree of organization in the tissue architecture has a bigger effect on local mechanical properties than the average mineral particle thickness. Similar results have been reported in previous nanomechanical (56) and sSAXS (44) analyses in the callus in a 3-mm fracture gap in sheep tibia.…”

“…The fact that no woven bone can be found adjacent to the scaffold surface supports this. This mode of scaffold-guided bone formation substantially differs from the two-wave theory that Liu and colleagues (44) suggested occurred in callus formation in a 3-mm fracture gap. Mineral particle thickness values in the deep cortex at 3 months agree with previous data.…”

“…Mineral particle thickness values in the deep cortex at 3 months agree with previous data. (44) Interestingly, in the newly formed bone, thicker mineral particles are found compared with those in the deep cortex, regardless of location and maturation time. The less compact collagen structure in the newly formed tissue might enable the growth of thicker particles.…”

“…A possible explanation could be that this tissue formed at a later time point. In regular fracture healing, Liu and colleagues (44) also observed faster growth of mineral particle thickness than increase in the r parameter; at 9 weeks the mineral particle thickness in the callus was larger than that in the cortex, although the r parameter was still much lower. Measurements of local elastic indentation modulus in the cortical bone after 12 months show lower values than previously reported data, (56) 20.8 AE 2.6 GPa as opposed to 28 GPa, probably because the latter was measured after 9 weeks in regions in the cortex, where no cortex remodeling had occurred yet.…”

“…(34,37,42,43) Lamellar bone is characterized by fibers with diameters of 2 mm to 3 mm arranged in lamellae parallel to the initial surface, lower mineral content, and osteocytes with ellipsoidal shape, with the longest axis aligned parallel to the fibers. (34,(41)(42)(43) Liu and colleagues (44) reported that the callus formation in a 3-mm osteotomy gap in the midshaft of a sheep tibia observed over 2 to 9 weeks occurred in two waves: As highly disordered woven bone was replaced by lamellar bone, the mineral particle size and degree of alignment increased with time, with spatial gradients at any given time point, depending on the radial and axial proximity to the cortex. As lamellar bone fills the woven bone cavities, primary osteons form with collagen fibers enclosing blood vessels embedded in a highly mineralized interstitial matrix.…”