Lactation-Induced Changes in the Volume of Osteocyte Lacunar-Canalicular Space Alter Mechanical Properties in Cortical Bone Tissue

Abstract

Osteocytes can remove and remodel small amounts of their surrounding bone matrix through osteocytic osteolysis, which results in increased volume occupied by lacunar and canalicular space (LCS). It is well established that cortical bone stiffness and strength are strongly and inversely correlated with vascular porosity, but whether changes in LCS volume caused by osteocytic osteolysis are large enough to affect bone mechanical properties is not known. In the current studies we tested the hypotheses that (1) lactation and postlactation recovery in mice alter the elastic modulus of bone tissue, and (2) such local changes in mechanical properties are related predominantly to alterations in lacunar and canalicular volume rather than bone matrix composition. Mechanical testing was performed using microindentation to measure modulus in regions containing solely osteocytes and no vascular porosity. Lactation caused a significant (∼13%) reduction in bone tissue-level elastic modulus (p < 0.001). After 1 week postweaning (recovery), bone modulus levels returned to control levels and did not change further after 4 weeks of recovery. LCS porosity tracked inversely with changes in cortical bone modulus. Lacunar and canalicular void space increased 7% and 15% with lactation, respectively (p < 0.05), then returned to control levels at 1 week after weaning. Neither bone mineralization (assessed by high-resolution backscattered scanning electron microscopy) nor mineral/matrix ratio or crystallinity (assessed by Raman microspectroscopy) changed with lactation. Thus, changes in bone mechanical properties induced by lactation and recovery appear to depend predominantly on changes in osteocyte LCS dimensions. Moreover, this study demonstrates that tissue-level cortical bone mechanical properties are rapidly and reversibly modulated by osteocytes in response to physiological challenge. These data point to a hitherto unappreciated role for osteocytes in modulating and maintaining local bone mechanical properties. © 2016 American Society for Bone and Mineral Research.

Keywords: LACTATION; LACUNAR-CANALICULAR SPACE; MECHANICAL PROPERTIES; OSTEOCYTE; OSTEOCYTIC OSTEOLYSIS; POROSITY.

Fig. 1

(A) Mouse femoral mid-diaphysis cross-section showing position of microindents and (B) enlarged scanning electron microscopy image shows a single residual indent profile.

Fig. 2

(A) BSEM image of the anterior mid-diaphysis cortex cross section of mouse femur (small arrowheads indicate osteocyte lacunae, longer white arrows show residual indents from mechanical testing). (B) BSEM image of longitudinal section through anterior femoral cortex, showing large numbers of canaliculi in cross-sectional view (black arrows) as well as an osteocyte lacuna (Lc) at the top of the image. (C) 3D Z-stack SIM image of osteocyte lacuna and canaliculi and (D) SIM visualization of canalicular lumena. Lc = lacuna.

Fig. 3

Elastic moduli (GPa) in the anterior (A) and posterior (B) mid-diaphyseal cortex of mouse femur following lactation and recovery, measure from microindentation testing. (p < 0.001 versus age-matched control bone).

Fig. 4

(A) Lacunar and (B) canalicular area (µm²) for control, lactation and 1-week recovery groups, determined using BSEM and SIM images, showing increases in void space with lactation and recovery to baseline within 1-week postlactation. (C) Lacunar, canalicular, and combined lacunar+canalicular porosity (BSEM studies) in control, lactation, and 1-week recovery bones, expressed as percentage of bone volume (p < 0.05 versus age-matched control).

Fig. 5

(A) BSEM image of longitudinal section through a mouse femoral diaphysis, showing canaliculi in cross-section. A single canaliculus with sampling overlay was used for radial distance measurement of mineralization. (B) Normalized mineralization plotted versus normalized distance from canalicular walls for control and lactation bones, showing no difference in matrix mineralization distribution profiles between groups. (Note: Offset in distances between treatment groups reflects effect of canalicular enlargement and for simplicity, data are shown from the canalicular wall to half the distance to adjacent canaliculi, as data beyond the midpoint are the mirror image of those shown).

Fig. 6

(A) Mineral/matrix ratio, (B) crystallinity, and (C) carbonate/phosphate ratio for control, lactation, and 1-week recovery bones obtained by Raman microspectroscopy. Lactation resulted in a significant (p < 0.05) carbonate loss that did not recover after cessation of lactation.