Michael Amling

Leptin Inhibits Bone Formation through a Hypothalamic Relay: A Central Control of Bone Mass

Gonadal failure induces bone loss while obesity prevents it. This raises the possibility that bone mass, body weight, and gonadal function are regulated by common pathways. To test this hypothesis, we studied leptin-deficient and leptin receptor-deficient mice that are obese and hypogonadic. Both mutant mice have an increased bone formation leading to high bone mass despite hypogonadism and hypercortisolism. This phenotype is dominant, independent of the presence of fat, and specific for the absence of leptin signaling. There is no leptin signaling in osteoblasts but intracerebroventricular infusion of leptin causes bone loss in leptin-deficient and wild-type mice. This study identifies leptin as a potent inhibitor of bone formation acting through the central nervous system and therefore describes the central nature of bone mass control and its disorders.

Rescue of the Skeletal Phenotype of Vitamin D Receptor- Ablated Mice in the Setting of Normal Mineral Ion Homeostasis: Formal Histomorphometric and Biomechanical Analyses*

1,25-Dihydroxyvitamin D3 has been shown to play an important role in vitro in regulating osteoblast gene transcription and promoting osteoclast differentiation. To address the role of the vitamin D receptor (VDR) in skeletal homeostasis, formal histomorphometric analyses were performed in VDR null mice in the setting of impaired mineral ion homeostasis as well as in VDR null mice in whom normal mineral ion homeostasis had been preserved. In hypocalcemic VDR null mice, there was an increase in bone volume as a result of a dramatic increase in osteoid. There was also an increase in the number of osteoblasts without a significant change in the number of osteoclasts. Examination of the growth plate revealed marked disorganization, with an increase in vascularity and matrix. Biomechanical parameters demonstrated increased bone fragility in the hypocalcemic VDR null mice. In the VDR ablated mice in whom normal mineral ion homeostasis had been preserved, none of these measurements was significantly different fro...

Bone mineralization defects and vitamin D deficiency: Histomorphometric analysis of iliac crest bone biopsies and circulating 25‐hydroxyvitamin D in 675 patients

Parathyroid hormone (PTH) is only one measurable index of skeletal health, and we reasoned that a histomorphometric analysis of iliac crest biopsies would be another and even more direct approach to assess bone health and address the required minimum 25‐Hydroxyvitamin D [25(OH)D] level. A cohort from the northern European population with its known high prevalence of vitamin D deficiency therefore would be ideal to answer the latter question. We examined 675 iliac crest biopsies from male and female individuals, excluding all patients who showed any signs of secondary bone diseases at autopsy. Structural histomorphometric parameters, including osteoid indices, were quantified using the Osteomeasure System according to ASBMR standards, and serum 25(OH)D levels were measured for all patients. Statistical analysis was performed by Students t test. The histologic results demonstrate an unexpected high prevalence of mineralization defects, that is, a pathologic increase in osteoid. Indeed, 36.15% of the analyzed patients presented with an osteoid surface per bone surface (OS/BS) of more than 20%. Based on the most conservative threshold that defines osteomalacia at the histomorphometric level with a pathologic increase in osteoid volume per bone volume (OV/BV) greater than 2% manifest mineralization defects were present in 25.63% of the patients. The latter were found independent of bone volume per trabecular volume (BV/TV) throughout all ages and affected both sexes equally. While we could not establish a minimum 25(OH)D level that was inevitably associated with mineralization defects, we did not find pathologic accumulation of osteoid in any patient with circulating 25(OH)D above 75 nmol/L. Our data demonstrate that pathologic mineralization defects of bone occur in patients with a serum 25(OH)D below 75 nmol/L and strongly argue that in conjunction with a sufficient calcium intake, the dose of vitamin D supplementation should ensure that circulating levels of 25(OH)D reach this minimum threshold (75 nmol/L or 30 ng/mL) to maintain skeletal health.

Genetic ablation of parathyroid glands reveals another source of parathyroid hormone.

The parathyroid glands are the only known source of circulating parathyroid hormone (PTH), which initiates an endocrine cascade that regulates serum calcium concentration. Glial cells missing2 (Gcm2), a mouse homologue of Drosophila Gcm, is the only transcription factor whose expression is restricted to the parathyroid glands. Here we show that Gcm2-deficient mice lack parathyroid glands and exhibit a biological hypoparathyroidism, identifying Gcm2 as a master regulatory gene of parathyroid gland development. Unlike PTH receptor-deficient mice, however, Gcm2-deficient mice are viable and fertile, and have only a mildly abnormal bone phenotype. Despite their lack of parathyroid glands, Gcm2-deficient mice have PTH serum levels identical to those of wild-type mice, as do parathyroidectomized wild-type animals. Expression and ablation studies identified the thymus, where Gcm1, another Gcm homologue, is expressed, as the additional, downregulatable source of PTH. Thus, Gcm2 deletion uncovers an auxiliary mechanism for the regulation of calcium homeostasis in the absence of parathyroid glands. We propose that this backup mechanism may be a general feature of endocrine regulation.

Overexpression of ΔFosB transcription factor(s) increases boneformation and inhibits adipogenesis

Members of the AP-1 family of transcription factors participate in the regulation of bone cell proliferation and differentiation. We report here a potent AP-1-related regulator of osteoblast function: ΔFosB, a naturally occurring truncated form of FosB that arises from alternative splicing of the fosB transcript and is expressed in osteoblasts. Overexpression of ΔFosB in transgenic mice leads to increased bone formation throughout the skeleton and a continuous post-developmental increase in bone mass, leading to osteosclerosis. In contrast, ΔFosB inhibits adipogenesis both in vivo and in vitro, and downregulates the expression of early markers of adipocyte differentiation. Because osteoblasts and adipocytes are thought to share a common precursor, it is concluded that ΔFosB transcriptionally regulates osteoblastogenesis, possibly at the expense of adipogenesis.

Modes of implant failure after hip resurfacing: morphological and wear analysis of 267 retrieval specimens.

BACKGROUND Resurfacing of the hip joint is experiencing a revival due to improvements in materials, design, and manufacturing techniques. Despite good midterm outcomes, the high early rate of failure and concerns about metal debris require a detailed morphological and wear analysis of retrieved resurfacing implants in order to understand failure mechanisms. METHODS A worldwide collection of hip resurfacing revision devices was initiated, and 267 components were received. Devices were analyzed by patient demographics, radiographic positioning, and wear, as well as morphologically and histologically. Specimens were grouped into four different failure types. They were also stratified into rim-loaded or non-rim-loaded groups. Failures were also assessed by surgeon learning-curve effects. RESULTS Time to failure was significantly different between the four revision-type groups: Specimens with fractures involving the implant rim were most common (46%) and failed earliest after surgery (mean of ninety-nine days), followed by fractures inside the femoral head (20%, 262 days) and loose cups (9%, 423 days). Revisions not due to fractures or cup loosening (25%) occurred at a mean of 722 days after surgery. Rim-loaded implants exhibited an average twenty-one to twenty-sevenfold higher wear rate than implants without rim-loading. Rim-loaded implants also showed a steeper mean cup inclination than their non-rim-loaded counterparts (59 degrees compared with 50 degrees ). Most failures occurred during the learning curve of the surgeon (the first fifty to 100 implantations). CONCLUSIONS Failures on the femoral side usually occur within the first nine months after surgery and appear to be most directly related to the implantation technique or patient selection. Later failures are observed mainly due to acetabular problems, either due to dramatically increased wear or poor cup anchorage. Improper cup anteversion may be similar to or more important than cup inclination in producing excessive wear.

Osteopenia and decreased bone formation in osteonectin-deficient mice

Bone continuously remodels in response to mechanical and physiological stresses, allowing vertebrates to renew bone as adults. Bone remodeling consists of the cycled synthesis and resorption of collagenous and noncollagenous extracellular matrix proteins, and an imbalance in this process can lead to disease states such as osteoporosis, or more rarely, osteopetrosis. There is evidence that the extracellular matrix glycoprotein osteonectin or secreted protein acidic and rich in cysteine (BM-40) may be important in bone remodeling. Osteonectin is abundant in bone and is expressed in areas of active remodeling outside the skeleton. In vitro studies indicate that osteonectin can bind collagen and regulate angiogenesis, metalloproteinase expression, cell proliferation, and cell-matrix interactions. In some osteopenic states, such as osteogenesis imperfecta and selected animal models for bone fragility, osteonectin expression is decreased. To determine the function of osteonectin in bone, we used contact x-ray, histomorphometry, and Northern blot analysis to characterize the skeletal phenotype of osteonectin-null mice. We found that osteonectin-null mice have decreased bone formation and decreased osteoblast and osteoclast surface and number, leading to decreased bone remodeling with a negative bone balance and causing profound osteopenia. These data indicate that osteonectin supports bone remodeling and the maintenance of bone mass in vertebrates.

Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind to and activate the same PTH/PTHrP receptor. Deletion of either the PTHrP gene or the PTH/PTHrP receptor gene leads to acceleration of differentiation of growth plate chondrocytes. To explore further the functional relationships of PTHrP and the PTH/PTHrP receptor, bones of knockout mice were analyzed early in development, and the phenotypes of double-knockout mice were characterized. One early phenotype is shared by both knockouts. Normally, the first chondrocytes to become hypertrophic are located in the centers of long bones; this polarity is greatly diminished in both these knockouts. The PTH/PTHrP receptor-deficient (PTH/PTHrP-R(-/-)) mice exhibited 2 unique phenotypes not shared by the PTHrP(-/-) mice. During intramembranous bone formation in the shafts of long bones, only the PTH/PTHrP-R(-/-) bones exhibit a striking increase in osteoblast number and matrix accumulation. Furthermore, the PTH/PTHrP-R(-/-) mice showed a dramatic decrease in trabecular bone formation in the primary spongiosa and a delay in vascular invasion of the early cartilage model. In the double-homozygous knockout mice, the delay in vascular invasion did not occur. Thus, PTHrP must slow vascular invasion by a mechanism independent of the PTH/PTHrP receptor.

Glucocorticoids Suppress Bone Formation by Attenuating Osteoblast Differentiation via the Monomeric Glucocorticoid Receptor

Development of osteoporosis severely complicates long-term glucocorticoid (GC) therapy. Using a Cre-transgenic mouse line, we now demonstrate that GCs are unable to repress bone formation in the absence of glucocorticoid receptor (GR) expression in osteoblasts as they become refractory to hormone-induced apoptosis, inhibition of proliferation, and differentiation. In contrast, GC treatment still reduces bone formation in mice carrying a mutation that only disrupts GR dimerization, resulting in bone loss in vivo, enhanced apoptosis, and suppressed differentiation in vitro. The inhibitory GC effects on osteoblasts can be explained by a mechanism involving suppression of cytokines, such as interleukin 11, via interaction of the monomeric GR with AP-1, but not NF-kappaB. Thus, GCs inhibit cytokines independent of GR dimerization and thereby attenuate osteoblast differentiation, which accounts, in part, for bone loss during GC therapy.

Increased bone mass is an unexpected phenotype associated with deletion of the calcitonin gene

Calcitonin (CT) is a known inhibitor of bone resorption. Calcitonin gene-related peptide-alpha (CGRPalpha), produced by alternative RNA processing of the CT/CGRP gene, has no clearly defined role in bone. To better understand the physiologic role of the CT/CGRP gene we created a mouse in which the coding sequences for both CT and CGRPalpha were deleted by homologous recombination. The CT/CGRP(-/-) knockout (KO) mice procreated normally, there were no identifiable developmental defects at birth, and they had normal baseline calcium-related chemistry values. However, KO animals were more responsive to exogenous human parathyroid hormone as evidenced by a greater increase of the serum calcium concentration and urine deoxypyridinoline crosslinks, an effect reversed by CT and mediated by a greater increase in bone resorption than in controls. Surprisingly, KO mice have significantly greater trabecular bone volume and a 1.5- to 2-fold increase in bone formation at 1 and 3 months of age. This effect appears to be mediated by increased bone formation. In addition, KO mice maintain bone mass following ovariectomy, whereas wild-type mice lose approximately one-third of their bone mass over 2 months. These findings argue for dual roles for CT/CGRP gene products: prevention of bone resorption in hypercalcemic states and a regulatory role in bone formation.