Bone cell-independent benefits of raloxifene on the skeleton: A novel mechanism for improving bone material properties

Abstract

Raloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (−OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle x-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength.

Structural features underlying raloxifene’s biophysical interaction with bone matrix

“…All animals were on protocols that were approved by the Institutional Animal Care and Use Committee prior to their use. Femora from skeletally mature (15-24 mo/old) treatment naïve male hounds were machined into prismatic beams (N = 8-12 beams per experimental group) following the details described previously [15]. Beams were subjected to at least one freeze-thaw cycle to ensure eradication of cellular activity.…”

“…Beams were subjected to at least one freeze-thaw cycle to ensure eradication of cellular activity. Absence of any cellular activity after a single freeze-thaw was demonstrated by lactate dehydrogenase immunostaining of fresh and frozen-thawed bone and the results have been published in [15]. Specified compounds were dissolved in dimethyl sulfoxide (DMSO) and then added to incubation media (1X PBS with 1% penicillin-streptomycin supplementation) at a 2 μM concentration.…”

“…Control bones were soaked in PBS with 0.04% DMSO vol/vol ratio. At the conclusion of soaking, bone beams were subjected to four-point bending as previously described [15]. Energy to fracture was measured as the area under the force displacement curve.…”

“…Preclinical studies have documented that raloxifene leads to improved mechanical properties, most notably material-level toughness, despite little/no change in BMD or geometry/architecture [12][13][14]. Recent work has demonstrated that this effect of raloxifene is at least partly cell-independent [15]. Mechanistically, it has been proposed that raloxifene increases bound water within the matrix and this alters how strains are transferred between the organic and hydroxyapatite (HAP) portions of the matrix.…”

Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils

“…Although both requirements have been alleviated with the advent of environmental SEM, which can image samples 'wet' and under moderate pressures and has thus enabled in situ experiments of biological samples, reported in situ SEM experiments have been very limited until now for bone tissue, because they currently cannot provide sufficient spatial resolution that would allow assessment of the bone ultrastructure organization. X-ray-based techniques such as SAXS and WAXS have been used in recent years for in situ experiments [149,150,152,[276][277][278][279]. However, these studies have been carried out for a limited number of discrete points in space within the sample only, and they could not provide quantitative results in terms of the 3D orientation of the ultrastructure.…”