Laleh Ardeshirpour

Demonstration of osteocytic perilacunar/canalicular remodeling in mice during lactation

Osteoclasts are thought to be solely responsible for the removal of bone matrix. However, we show here that osteocytes can also remove bone matrix by reversibly remodeling their perilacunar/canalicular matrix during the reproductive cycle. In contrast, no osteocytic remodeling was observed with experimental unloading despite similar degrees of bone loss. Gene array analysis of osteocytes from lactating animals revealed an elevation of genes known to be utilized by osteoclasts to remove bone, including tartrate‐resistant acid phosphatase (TRAP) and cathepsin K, that returned to virgin levels upon weaning. Infusion of parathyroid hormone–related peptide (PTHrP), known to be elevated during lactation, induced TRAP activity and cathepsin K expression in osteocytes concurrent with osteocytic remodeling. Conversely, animals lacking the parathyroid hormone type 1 receptor (PTHR1) in osteocytes failed to express TRAP or cathepsin K or to remodel their osteocyte perilacunar matrix during lactation. These studies show that osteocytes remove mineralized matrix through molecular mechanisms similar to those utilized by osteoclasts.

Site‐specific changes in bone microarchitecture, mineralization, and stiffness during lactation and after weaning in mice

Despite the dramatic bone loss that occurs during lactation, bone mineral density rapidly recovers after offspring are weaned and milk production stops. The goal of this study is to quantify site‐specific changes in bone quantity and quality during and after lactation in a mouse model. We used micro computed tomography (µCT), individual trabecula segmentation (ITS), digital topological analysis (DTA)‐based tissue mineral density (TMD) analysis, and micro finite element analysis (µFEA) to quantify the effects of lactation and weaning on bone microarchitecture, mineralization, and stiffness at the spine, tibia, and femur. We found a significant decrease in trabecular plate microarchitecture, tissue mineralization of the trabecular surface, trabecular central skeleton, and intervening envelopes, and whole bone stiffness in lactating versus nulliparous mice at all three sites. In recovered mice, all these different aspects of bone quality were comparable to nulliparous mice at the spine. In contrast, trabecular plate microarchitecture and whole bone stiffness at the tibia and femur in recovered mice were lower than nulliparous mice, as were central trabecular tissue mineralization and cortical structure at the femur. These findings are consistent with clinical observations of partial recovery of femoral bone mineral density BMD after lactation in humans. The observed differences in trabecular surface tissue mineralization in nulliparous, lactating, and recovered mice are consistent with prior observations that maternal bone turnover shifts from resorption to formation at the time of pup weaning. The significant differences in trabecular central tissue mineralization during these three states suggest that osteocytes may contribute to the reversible loss of mineral during and after lactation. Future studies are necessary to determine whether differing functions of various bone cells at individual skeletal sites cause site‐specific skeletal changes during and after lactation.

Increased PTHrP and decreased estrogens alter bone turnover but do not reproduce the full effects of lactation on the skeleton.

During lactation, calcium is mobilized from the maternal skeleton to supply the breast for milk production. This results in rapid but fully reversible bone loss. Prior studies have suggested that PTHrP, secreted from the breast, and estrogen deficiency, due to suckling-induced central hypogonadism, combine to trigger bone resorption. To determine whether this combination was sufficient to explain bone loss during lactation, we raised PTHrP levels and decreased levels of estrogens in nulliparous mice. PTHrP was infused via osmotic minipumps and estrogens were decreased either by using leuprolide, a long-acting GnRH agonist, or by surgical ovariectomy (OVX). Bone mineral density declined by 23.2 ± 1.3% in the spine and 16.8 ± 1.9% in the femur over 10 d of lactation. This was accompanied by changes in trabecular architecture and an increase in both osteoblast and osteoclast numbers. OVX and PTHrP infusion both induced a modest decline in bone mineral density over 10 d, but leuprolide treatment did not. The combination of OVX and PTHrP was more effective than either treatment alone, but there was no interaction between PTHrP and leuprolide. None of the treatments reproduced the same degree of bone loss caused by lactation. However, both forms of estrogen deficiency led to an increase in osteoclasts, whereas infusion of PTHrP increased both osteoblasts and osteoclasts. Therefore, although the combination of PTHrP and estrogen deficiency contributes to bone loss, it is insufficient to reproduce the full response of the skeleton to lactation, suggesting that other factors also regulate bone metabolism during this period.

Skeletal recovery after weaning does not require PTHrP.

Mice lose 20% to 25% of trabecular bone mineral content (BMC) during lactation and restore it after weaning through unknown mechanisms. We found that tibial Pthrp mRNA expression was upregulated fivefold by 7 days after weaning versus end of lactation in wild‐type (WT) mice. To determine whether parathyroid hormone–related protein (PTHrP) stimulates bone formation after weaning, we studied a conditional knockout in which PTHrP is deleted from preosteoblasts and osteoblasts by collagen I promoter–driven Cre (CreColI). These mice are osteopenic as adults but have normal serum calcium, calcitriol, and parathyroid hormone (PTH). Pairs of Pthrpflox/flox;CreColI (null) and WT;CreColI (WT) females were mated and studied through pregnancy, lactation, and 3 weeks of postweaning recovery. By end of lactation, both genotypes lost lumbar spine BMC: WT declined by 20.6% ± 3.3%, and null decreased by 22.5% ± 3.5% (p < .0001 versus baseline; p = NS between genotypes). During postweaning recovery, both restored BMC to baseline: WT to –3.6% ± 3.7% and null to 0.3% ± 3.7% (p = NS versus baseline or between genotypes). Similar loss and full recovery of BMC were seen at the whole body and hind limb. Histomorphometry confirmed that nulls had lower bone mass at baseline and that this was equal to the value achieved after weaning. Osteocalcin, propeptide of type 1 collagen (P1NP), and deoxypyridinoline increased equally during recovery in WT and null mice; PTH decreased and calcitriol increased equally; serum calcium was unchanged. Urine calcium increased during recovery but remained no different between genotypes. Although osteoblast‐derived PTHrP is required to maintain adult bone mass and Pthrp mRNA upregulates in bone after weaning, it is not required for recovery of bone mass after lactation. The factors that stimulate postweaning bone formation remain unknown.

A double-blind, placebo-controlled comparison of letrozole to oxandrolone effects upon growth and puberty of children with constitutional delay of puberty and idiopathic short stature.

Background/Aims: Constitutional delay of growth and puberty (CDGP) with short stature is one of the most common problems in pediatrics. We compared the effects of letrozole with that of oxandrolone on predicted adult height (PAH), puberty, bone mineral density, serum insulin-like growth factor 1 (IGF-1) and blood lipoproteins. Methods: In a prospective, double-blind, randomized, placebo-controlled clinical trial, 91 CDGP boys (12.6–14.6 years old) with predicted short stature were treated with letrozole (2.5 mg/day), oxandrolone (2.5 mg/day), or placebo, at the outpatient pediatric endocrine clinic of Mofid Children’s Hospital in Tehran for 2 years. Results: Letrozole differed from oxandrolone and placebo in significantly increasing PAH (p < 0.05), and slightly but significantly decreasing HDL-cholesterol. Oxandrolone, and to a lesser degree letrozole, significantly increased the height standard deviation score and bone age compared to placebo. Conclusion: This first randomized controlled clinical trial in CDGD teenage boys with predicted short stature shows that letrozole increases PAH more than oxandrolone and advances pubertal stage and bone mineralization less.

OPG Treatment Prevents Bone Loss During Lactation But Does Not Affect Milk Production or Maternal Calcium Metabolism

Lactation is associated with increased bone turnover and rapid bone loss, which liberates skeletal calcium used for milk production. Previous studies suggested that an increase in the skeletal expression of receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand (RANKL) coupled with a decrease in osteoprotegerin (OPG) levels likely triggered bone loss during lactation. In this study, we treated lactating mice with recombinant OPG to determine whether bone loss during lactation was dependent on RANKL signaling and whether resorption of the maternal skeleton was required to support milk production. OPG treatment lowered bone resorption rates and completely prevented bone loss during lactation but, surprisingly, did not decrease osteoclast numbers. In contrast, OPG was quite effective at lowering osteoblast numbers and inhibiting bone formation in lactating mice. Furthermore, treatment with OPG during lactation prevented the usual anabolic response associated with reversal of lactational bone loss after weaning. Preventing bone loss had no appreciable effect on milk production, milk calcium levels, or maternal calcium homeostasis when mice were on a standard diet. However, when dietary calcium was restricted, treatment with OPG caused maternal hypocalcemia, maternal death, and decreased milk production. These studies demonstrate that RANKL signaling is a requirement for bone loss during lactation, and suggest that osteoclast activity may be required to increase osteoblast numbers during lactation in preparation for the recovery of bone mass after weaning. These data also demonstrate that maternal bone loss is not absolutely required to supply calcium for milk production unless dietary calcium intake is inadequate.