Pia Pogoda

Leptin Inhibits Bone Formation Not Only in Rodents, but Also in Sheep

This study examines the effect of long‐term ICV administration of leptin in ewes. We found that central application significantly decreased osteoblast activity as measured by serum analysis as well as by histomorphometry, resulting in decreased trabecular bone volume. These data provide additional evidence that bone formation and therefore bone remodeling is at least in part centrally controlled.

Bone remodeling: new aspects of a key process that controls skeletal maintenance and repair

Bone remodeling is the concerted interplay of two cellular activities: osteoclastic bone resorption and osteoblastic bone formation. Bone remodeling is the physiologic process that maintains bone mass, skeletal integrity and skeletal function. A molecular understanding of this process is therefore of paramount importance for almost all aspects of skeletal physiology and many facets of bone diseases. Based on the morphological observation of the BMU—”bone multicellular unit” or “bone metabolic unit”—and a wide body of in vitro data, bone remodeling was thought to be controlled locally through functional coupling of resorption and formation and vice versa. However, recent genetic studies have shown that there is no obligatory tight cross-control of bone formation and bone resorption in vivo and that there is also a central axis controlling bone formation, one aspect of bone remodeling. The molecule that inhibits bone formation through a hypothalamic relay is leptin. Following binding to its receptor located on the ventromedial nuclei of the hypothalamus, leptin’s action on bone formation is mediated via a neuronal signaling cascade that involves the ß-adrenergic system. The overall goal of this review is to show how the dialogue between clinical medicine and mouse genetics helped to uncover a new concept in skeletal physiology.

Age- and sex-related changes of humeral head microarchitecture: Histomorphometric analysis of 60 human specimens†

Fractures of the humeral head are frequent and will further increase due to demographic changes. Prior to operative fracture treatment, the regional differences of bone quality, especially of elderly people, have to be carefully considered to assure stable implant fixation. However, conclusive data concerning the variation of histomorphometric parameters are still lacking. Consequently, the purpose of this study was to analyze the age‐ and sex‐related changes in bone microarchitecture. For that reason, 60 proximal humeri were harvested from patients at autopsy. Twelve regions of interest (ROI) were defined for each centered coronar humeral head slice and the specimens were subjected to radiographic, histological, and histomorphometric analyses. We could demonstrate that in contrast to men, women over 60 years of age had a significant age‐related decrease in bone mass. The most prominent decrease was observed in the region of the greater tuberosity, which represents an osteoporotic fracture site. The most superior and medially located part of the centered coronar humeral head slice showed, independent from age and sex, the highest bone mass and can therefore be considered as the best location for subchondral screw placement. Taken together, our study revealed distinct sex‐related changes of the humeral head bone microarchitecture with aging, which should be considered in implant positioning.

Skeletal mineralization defects in adult hypophosphatasia—a clinical and histological analysis

SummaryHistomorphometry and quantitative backscattered electron microscopy of iliac crest biopsies from patients with adult hypophosphatasia not only confirmed the expected enrichment of non-mineralized osteoid, but also demonstrated an altered trabecular microarchitecture, an increased number of osteoblasts, and an impaired calcium distribution within the mineralized bone matrix.IntroductionAdult hypophosphatasia is an inherited disorder of bone metabolism caused by inactivating mutations of the ALPL gene, encoding tissue non-specific alkaline phosphatase. While it is commonly accepted that the increased fracture risk of the patients is the consequence of osteomalacia, there are only few studies describing a complete histomorphometric analysis of bone biopsies from affected individuals. Therefore, we analyzed iliac crest biopsies from eight patients and set them in direct comparison to biopsies from healthy donors or from individuals with other types of osteomalacia.MethodsHistomorphometric analysis was performed on non-decalcified sections stained either after von Kossa/van Gieson or with toluidine blue. Bone mineral density distribution was quantified by backscattered electron microscopy.ResultsBesides the well-documented enrichment of non-mineralized bone matrix in individuals suffering from adult hypophosphatasia, our histomorphometric analysis revealed alterations of the trabecular microarchitecture and an increased number of osteoblasts compared to healthy controls or to individuals with other types of osteomalacia. Moreover, the analysis of the mineralized bone matrix revealed significantly decreased calcium content in patients with adult hypophosphatasia.ConclusionsTaken together, our data show that adult hypophosphatasia does not solely result in an enrichment of osteoid, but also in a considerable degradation of bone quality, which might contribute to the increased fracture risk of the affected individuals.

Subdental synchondrosis and anatomy of the axis in aging: a histomorphometric study on 30 autopsy cases

During skeletal development the two ossification centers of the odontoid process are separated from the corpus of the axis by a subdental synchondrosis. This synchondrosis is thought to close and disappear spontaneously in adolescence although this has never been studied in detail. The basis of the dens is of clinical relevance as type II dens fractures are located here. To characterize the morphological architecture of the axis with particular attention to the subdental synchondrosis, the complete axis was harvested from thirty age-matched and gender-matched patients of the three different age groups at autopsy. The subdental synchondrosis and the bone structure of the dens, the basis of the dens and the body of C2 were analyzed by radiography, histology and quantitative histomorphometry. At the macroscopic level the persistency of the subdental synchondrosis in the adult cervical spine was detected in 87% (26 of 30) of the specimens. Histomorphometry revealed a residual disc blastema with an average size of 25.8% of the sagittal depth of the basis of the dens at this level. Bony integration of the synchondrosis was poor throughout all ages. Histologically a cartilaginous matrix composition of the subdental synchondrosis persisted throughout all groups. The trabecular microarchitecture demonstrated a significant reduction of bone volume and trabecular number as well as an increased trabecular separation within the basis of the dens as compared to the corpus or the dens of C2. This histomorphometric data regarding a poor integration of the synchondrosis into the trabecular network and the reduced bone mass within the basis of the dens might offer a previously underestimated explanation for the occurrence of type II dens fractures and their association with pseudoarthrosis, respectively.

Effects of Estrogen on Fracture Healing in Mice

BACKGROUND Fracture healing is a complex and sequential process. One important step in fracture healing is callus remodeling. Estrogen deficiency is known to increase osteoclast bone resorption, whereas estrogen replacement can reverse this effect. Therefore, the aim of our study was to analyze whether estrogen deficiency and estrogen treatment, respectively, would affect callus remodeling in the fracture healing process. METHODS Standardized femoral fractures were produced in 10 weeks old C57BL/6 mice using a guillotine-like fracture device. Mice were separated into three groups. The first group obtained a continuous administration of estrogen. Ovariectomy (OVX) was performed in the second group to generate an estrogen-deficiency model. The control group obtained no special treatment. At different stages of fracture healing, contact X-ray, micro-computed tomography, histologic, and biomechanical analyses were performed. RESULTS We observed that, in early stages of fracture healing, OVX leads to an impaired periosteal callus formation. When compared with the control group, chondrocytes area was decreased, and the subsequent mineralization was less distinctive. In the late stage of fracture healing, the OVX mice showed a thin and porous cortex. In sharp contrast, estrogen treatment led to an enhanced fracture healing. Chondrocyte areas were larger, callus mineralization was increased, and the neocortex was thicker. Biomechanical testing confirmed the beneficial effects of estrogen on restoration of biomechanical competence. CONCLUSION These results indicate that estrogen seems to be an important factor in all stages of fracture healing. The application of estrogens enhances fracture healing of long bones at least in mice.

Central Control of Bone Mass: Brainstorming of the Skeleton

Our understanding of the biology of the skeleton, like that of virtually every other subject in biology, has been transformed by recent advances in human and mouse genetics. These advances, together with findings from the work of chicken embryologists, have radically enhanced our comprehension of the developmental biology of the vertebrate skeleton.1,2In contrast, we have learned very little, in molecular and genetic terms, about another very important part of skeletal biology, namely its physiology. Among the many questions of skeletal physiology that are largely unexplained are the following: Why and how do we stop growing? Why and how are bone and teeth the only organs to mineralize under physiological conditions? How is bone mass maintained nearly constant between the end of puberty and the arrest of gonadal functions? This review will deal with this last issue.

Mouse models in skeletal physiology and osteoporosis: experiences and data on 14839 cases from the Hamburg Mouse Archives

Our understanding of the developmental biology of the skeleton, like that of virtually every other subject in biology, has been transformed by recent advances in human and mouse genetics, but we still know very little, in molecular and genetic terms, about skeletal physiology. Thus, among the many questions that are largely unexplained are the following: why is osteoporosis mainly a women’s disease? How is bone mass maintained nearly constant between the end of puberty and the arrest of gonadal functions? Molecular genetics has emerged as a powerful tool to study previously unexplored aspects of the physiology of the skeleton. Among mammals, mice are the most promising animals for this experimental work. The input that transgenic animals can offer to our field depends on our means of phenotypic characterization of the mouse skeleton. In fact, full appreciation of the skeletal characteristics of a given mouse model requires the application of standardized protocols for noninvasive imaging, histology, histomorphometry, biomechanics, and individually adapted in vitro and in vivo analysis. Over the past years we have established a mouse archive that consists of 14839 cases from more than 120 different mouse models that we have phenotypically characterized in Hamburg. Today, this is one of the biggest databases on the mouse skeleton. This review focuses on one aspect of skeletal physiology, namely skeletal aging, and demonstrates that mouse models can be a valuable tool to gain insights in certain facets of skeletal physiology that have been unexplored previously.

Low turnover osteoporosis in sheep induced by hypothalamic-pituitary disconnection

The hypothalamus is of critical importance in regulating bone remodeling. This is underscored by the fact that intracerebroventricular‐application of leptin in ewe leads to osteopenia. As a large animal model of osteoporosis, this approach has some limitations, such as high technical expenditure and running costs. Therefore we asked if a surgical ablation of the leptin signaling axis would have the same effects and would thereby be a more useful model. We analyzed the bone phenotype of ewe after surgical hypothalamo‐pituitary disconnection (HPD + OVX) as compared to control ewe (OVX) after 3 and 12 months. Analyses included histomorphometric characterization, micro‐CT and measurement of bone turnover parameters. Already 3 months after HPD we found osteopenic ewe with a significantly decreased bone formation (69%) and osteoclast activity (49%). After a period of 12 months the HPD group additionally developed an (preclinical) osteoporosis with significant reduction (33%) of femoral cortical thickness, as compared to controls (OVX). Taken together, HPD leads after 12 month to osteoporosis with a reduction in both trabecular and cortical bone caused by a low bone turnover situation, with reduced osteoblast and osteoclast activity, as compared to controls (OVX). The HPD‐sheep is a suitable large animal model of osteoporosis. Furthermore our results indicate that an intact hypothalamo‐pituitary axis is required for activation of bone turnover.

High fluoride and low calcium levels in drinking water is associated with low bone mass, reduced bone quality and fragility fractures in sheep.

SummaryChronic environmental fluoride exposure under calcium stress causes fragility fractures due to osteoporosis and bone quality deterioration, at least in sheep. Proof of skeletal fluorosis, presenting without increased bone density, calls for a review of fracture incidence in areas with fluoridated groundwater, including an analysis of patients with low bone mass.IntroductionUnderstanding the skeletal effects of environmental fluoride exposure especially under calcium stress remains an unmet need of critical importance. Therefore, we studied the skeletal phenotype of sheep chronically exposed to highly fluoridated water in the Kalahari Desert, where livestock is known to present with fragility fractures.MethodsDorper ewes from two flocks in Namibia were studied. Chemical analyses of water, blood and urine were executed for both cohorts. Skeletal phenotyping comprised micro-computer tomography (μCT), histological, histomorphometric, biomechanical, quantitative backscattered electron imaging (qBEI) and energy-dispersive X-ray (EDX) analysis. Analysis was performed in direct comparison with undecalcified human iliac crest bone biopsies of patients with fluoride-induced osteopathy.ResultsThe fluoride content of water, blood and urine was significantly elevated in the Kalahari group compared to the control. Surprisingly, a significant decrease in both cortical and trabecular bones was found in sheep chronically exposed to fluoride. Furthermore, osteoid parameters and the degree and heterogeneity of mineralization were increased. The latter findings are reminiscent of those found in osteoporotic patients with treatment-induced fluorosis. Mechanical testing revealed a significant decrease in the bending strength, concurrent with the clinical observation of fragility fractures in sheep within an area of environmental fluoride exposure.ConclusionsOur data suggest that fluoride exposure with concomitant calcium deficit (i) may aggravate bone loss via reductions in mineralized trabecular and cortical bone mass and (ii) can cause fragility fractures and (iii) that the prevalence of skeletal fluorosis especially due to groundwater exposure should be reviewed in many areas of the world as low bone mass alone does not exclude fluorosis.