Morphogenesis of Floating Bone Segments: A Legacy of Serial Tensile Cross-Strut Microdamage in Trabecular Disconnection “Crumple Zones”?

Trabecular bone disconnection “hotspots” of real termini (ReTm) previously mapped as loci of weakness in the female aging spine and hip may be a source of free-floating cancellous segments found in the medullary space using a bespoke, thick slice histological method for identifying ReTm. A factor in their origin is apparently microdamage proliferation (differentiated by en bloc silver staining) with occasional callus moderation. Validation of similar “floating segments” (FS) in the ex-breeder rat suggested a pilot model for a potentially common phenomenon. Following marrow elution and density fractionation of the isolated floating segments from the whole proximal rat femora, scanning electron microscopy (SEM) and elemental microanalysis (EDS) was performed. The eluent contained numbers of vertically truncated, laterally branched floating segments (acute severance of sequential tensile cross-struts, causing chronic compression overload of axial-struts, with ii) inadequate stabilising callus, facilitating ReTm stacking into predetermined, substructural “crumple zones” of force containment, spheroidal attrition and particulate dissociation. As a catabolic outcome of altered tensile and hormonal influence, FS number may add a novel variable to cancellous bone kinetics particularly in women of relevance to fracture predisposition.

Mineral Fabrication and Golgi Apparatus Activity in <i>Spirostomum ambiguum</i>: A Primordial Paradigm of the Stressed Bone Cell?

The histological basis for acute osteocyte mechanosensitivity remains uncertain. A novel bone cell model of mechanotransduction and inorganic trafficking may be the powerful, silt-burrowing protozoan Spirostomum ambiguum which when being physically challenged fabricates within vesicles populations of bone-like calcium phosphate microspheres, about 1 μm in diameter. These not only attribute considerable compression-resilience but also resemble the Golgi-directed mineral assemblies we recently reported in osteocytes. Advantageously, calcification in the protozoan (confirmed by ultramicroscopy with EDX elemental microanalysis) enabled Golgi comparison under overt, natural phases of both high (i.e. silt-tunnelling) and low (i.e free-swimming) stress. Established hard-tissue microscopy techniques previously positive in bone cells included quantitative fluorescent tetracycline labelling for bone salt together with the same metazoan Golgi body marker (Green Fluorescent Protein-tagged mannosidase II construct). Organellar modulation was monitored by transfection of live organisms in situ (some post-stained with red nuclear fluorochrome TOPRO-3). Results showed that GFP-tagged Golgi fluorescence increased from swimmers (mean 74.5 ± SD 6.7 AU) to burrowers (mean 104.6 ± SD 2.7;p < 0.0001) synchronous with juxtanuclear tetracycline-labelled mineral fluorescence (swimmers, mean 89.7 ± SD 3.3 AU;burrowers, mean 138.0 ± SD 4.0;p < 0.0001). Intracellular dense microspheres, single or bridged, were harvested as pellets rich in Ca, P (Ca:P 0.98) and Si, their polarised alignment moving from transaxial in swimmers to axial in burrowers. It was concluded that Golgi-directed mineral fabrication in the large, accessible, silt-enclosed ciliate resembles that in the smaller, less-accessible bone cell and may be a conserved early mechanobiological intracellular development predicating force translation into compression-resistant mineral fabrication in loaded segments of the osteocyte syncitium.

Bone Mineral “Quality”: Differing Characteristics of Calcified Microsphere Populations at the Osteoporotic and Osteoarthritic Femoral Articulation Front

The mineral front consists of large populations of organically enshrouded calcified microspheres (filamentous clusters) about 1 micron in diameter and associated smaller numbers of variably dense nanospheres, 30 - 40 nm in diameter. The discrete objects persist and modulate in maturity, and may constitute a variable “qualitative” factor in the skeletal inorganic phase, exemplified by the biomechanically contrasting pathologies of osteoporosis (OP;fracture, low stress condition) and osteoarthritis (OA;non fracture, high stress condition). The aim was to compare the articulation front material for morphological and trace element diversity using fresh female femoral head discards (from Dewsbury District Hospital NHS Mid-Yorkshire Trust). These were prepared for histology of the cartilage/bone interface region by immersion in hydrazine hydrate to expose the anorganic mineral topography for microscopy and FEGSEM microanalysis. 1) Mineral microsphere morphology (compared to animals as arbitrary controls) suggested that calcified microspheres from OP (n = 19) tended to be small (0.5 - 0.7 microns), smooth and compacted;those from OA (n = 19) were large (0.5 - 4.0 microns), uneven and irregularly dispersed. Respective calcified nanospheres from OP were similarly smaller (30 - 50 nm) than those from OA (>100 nm). In subchondral bone a proportion of the filamentous microspheres had fused into a fine-textured phase in OP and a coarse-textured phase in OA. 2) Trace element analysis (compared to positive porcine Si and Mg and other peaks) suggested a diminution with mineral maturity, and also with age effecting OP and OA similarly, with minor differences detected between them. It was concluded that calcified objects constituting the inorganic phase vary regionally with age and in fracture and nonfracture, being diminished in size (not number) in weak OP and enlarged (compared to porcine control) in stronger OA, with Si or Mg “doping” diminishing with time, perhaps influencing their individual bioactivity and matrix dynamics.

Mineral Fabrication and Golgi Apparatus Activity in the Mouse Calvarium

There is diverse opinion about the mechanism of bone mineralization with only intermittent reports of any direct organellar role played by the golgi apparatus (juxtanuclear body). Light and laser confocal microscopy was combined with electron microscopy and elemental EDX (energy dispersive X-ray microanalysis) in comparing “young” osteocytes in situ in fresh and “slam” frozen developing mouse calvarium, with similar cells (MC3T3-E1) maintained in vitro. The distribution of “nascent” electron dense mineral was examined histochemically (von Kossa, GBHA), including tetracycline (TC) staining as a fluorescent complex with bone salt, while golgi body activity was demonstrated by transfection with a specific green fluorescent construct (GFP/mannosidase II). In tissue culture golgi body activity and mineralization were both blocked by brefeldin A (an established golgi inhibitor) and restored by forskolin, enabling an association with mineral fabrication to be quantified as changing fluorescence intensity (AU) of GFP or TC markers. Results from osteocytes in situ supported previous descriptions of intracellular electron dense objects (microspheres and nanospheres) in a juxtanuclear pattern, containing Ca, P and transitory Si, in a spectrum recapitulated in the calcifying culture after 10 days, when GFP fluorophore surged from 71.7 ± 1.4SD to 133.7 ± 2.7SD AU by 14 days (p < 0.0001). At this stage TC fluorophore mean intensity was 23.8 ± 3.7SD AU (14 days) rising to 45.0 ± 5.1SD AU by 17 days, compared to its stationary 21.7 ± 3.6SD when treated 3 days previously with BFA golgi inhibitor (p < 0.0001), until forskolin reversal. It was concluded from the changing juxtanuclear morphology, Si mineralization mediation and the variably controlled activity versus stasis that the inorganic phase of bone is a complex golgi-directed fabrication with implications for bone matrix biology and evolution.