Frank Timo Beil

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.

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.

Metaphyseal fracture healing follows similar biomechanical rules as diaphyseal healing

It is generally supposed that the pattern of fracture healing in trabecular metaphyseal bone differs from that of diaphyseal fractures. However, few experimental studies to date have been performed, even though clinically many fractures occur in metaphyseal bone. Particularly, the influence of biomechanical factors has not yet been investigated under standardized conditions. Our aim was to correlate the interfragmentary strain (IFS) with the bone‐healing outcome in a controlled metaphyseal fracture model in sheep. Twelve sheep received a partial osteotomy in the distal femoral condyle close to the trochlea. The determination of the IFS by in vivo X‐ray analyses and a finite element model revealed that the deflection of the osteotomy gap by the patello‐femoral force during walking provoked increasing strains of up to 40%. Bone healing was evaluated after 8 weeks by the assessment of the bone mineral density and by histomorphometry in regions of interest that displayed differing magnitudes of IFS. In areas with strains below 5% significantly less bone formation occurred compared to areas with higher strains (6–20%). For strains larger than 20% fibrocartilage layers were observed. Low IFS (<5%) led to intramembranous bone formation, whereas higher strains additionally provoked endochondral ossification or fibrocartilage formation. It is therefore proposed that metaphyseal bone healing follows similar biomechanical principles as diaphyseal healing.

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.

Biomechanical analysis of atlas fractures: a study on 40 human atlas specimens.

Study Design. Forty isolated specimens of the first cervical vertebra were tested by the application of pure axial force to failure. To exclude ligamentous side effects, transverse ligaments were dissected in all specimens. Objective. To investigate the biomechanical characteristics of the human atlas and to describe the influence of different speeds of force impact on the fracture types. Summary of Background Data. Atlas fractures have been reproduced in some studies in the literature. However, the characteristics of isolated atlas fractures under pure axial loading at different speeds has not been reported so far. Methods. After dissection of soft tissue and generation of a peripheral quantitative computed tomography scan, the atlas preparations were tested to failure by displacement-controlled axial force application at constant speeds of either 0.5 mm/s (Group 1) or 300 mm/s (Group 2). The fracture types were classified according to Gehweiler. Results. At slow loading speed (Group 1), 2 Type-I (anterior arch), 3 Type-II (posterior arch), 2 Type-III (anterior and posterior arch), and 13 Type-IV (lateral mass) fractures occurred out of 20 specimens. At high loading speed (Group 2), Type-III fractures (burst fractures of 2 to 4 parts) occurred in all 20 tested specimens. Conclusion. The presented results strongly suggest that the Type of atlas fracture depends on the speed of axial force impact. The present study demonstrates that Type-III fractures (2- to 4-part burst fractures) result from fast force impact whereas slow force impact is responsible for Type-IV atlas fractures of the lateral mass.

Sheep model for osteoporosis: Sustainability and biomechanical relevance of low turnover osteoporosis induced by hypothalamic–pituitary disconnection

Hypothalamo‐pituitary disconnection (HPD) leads to low bone turnover and osteoporosis in sheep. To determine the sustainability of bone loss and its biomechanical relevance, we studied HPD‐sheep 24 months after surgery (HPD + OVX‐24) in comparison to untreated control (Control), ovariectomized sheep (OVX), and sheep 12 months after HPD (HPD + OVX‐12). We performed histomorphometric, HR‐pQCT, and qBEI analyses, as well as biomechanical testing of all ewes studied. Twenty‐four months after HPD, histomorphometric analyses of the iliac crest showed a significant reduction of BV/TV by 60% in comparison to Control. Cortical thickness of the femora measured by HR‐pQCT did not change between 12 and 24 months after HPD but remained decreased by 30%. These structural changes were caused by a persisting depression of osteoblast and osteoclast cellular activity. Biomechanical testing of the femora showed a significant reduction of bending strength, whereas calcium content and distribution was found to be unchanged. In conclusion, HPD surgery leads to a persisting low turnover status with negative turnover balance in sheep followed by dramatic cortical and trabecular bone loss with consequent biomechanical impairment.