G. Delling

Loss of the ClC-7 Chloride Channel Leads to Osteopetrosis in Mice and Man

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.

Trabecular bone pattern factor: a new parameter for simple quantification of bone microarchitecture

The stability of trabecular bone depends not only on the amount of bone tissue, but also on the three-dimensional orientation and connectedness of trabeculae, which is summarized as trabecular microarchitecture. In previous studies we could demonstrate that in three-dimensional bone tissue the relation of trabecular plates to rods is reflected in the ratio of concave to convex surfaces of the bone pattern in two-dimensional bone sections. For the quantification of the connectedness of these bone patterns we developed a new histomorphometric parameter called Trabecular Bone Pattern factor (TBPf). The basic idea is that the connectedness of structures can be described by the relation of convex to concave surfaces. A lot of concave surfaces represent a well connected spongy lattice, whereas a lot of convex surfaces indicate a badly connected trabecular lattice in two-dimensional sections. By means of an automatic image analysis system we measure trabecular bone area (A1) and perimeter (P1). A second measurement of these two parameters (now A2 and P2) is done after a simulated dilatation of trabeculae on the screen. This dilatation results in a characteristic change of bone area and perimeter depending on the relation of convex to concave surfaces. TBPf is defined as a quotient of the difference of the first and the second measurement: TBPf = (P1 - P2)/(A1 - A2). First measurements of TBPf in 192 iliac crest bone biopsies of autopsy cases show that there is not only age-related loss of bone volume, but also a decrease of trabecular connectedness. By means of TBPf we can demonstrate a significant difference in the age-related loss of trabecular connectivity between male and female individuals.

Morphometric analysis of noninvasively assessed bone biopsies: Comparison of high-resolution computed tomography and histologic sections

This article describes a new method to analyze the structural behavior of trabecular bone structure by means of noninvasive measurements of the three-dimensional cancellous bone architecture. For the noninvasive data acquisition, a high-resolution quantitative computed tomography system for peripheral measuring sites (pQCT) was used. With this system and the help of a multiple thin-slice measuring technique, it became possible to examine three-dimensional bone structure with a resolution of 0.25 mm. Using a special three-dimensional segmentation algorithm, mineralized bone was separated from bone marrow and muscle tissue within the three-dimensional stack of CT slices. These segmented data sets can then be processed nondestructively and, even more important, repetitively in either two or three dimensions. In order to validate this noninvasive procedure, a two-dimensional comparative morphometric study was performed including CT slices and corresponding histologic sections prepared after CT measurement. Three representative sections from the three-dimensional stack of CT slices were selected and the morphometric indices of the segmented CT slices were compared with the indices stemming from the corresponding histologic sections prepared after CT measurement. Although the presented approach can only give an example of the method, the results from the morphometric analysis support the assumption that cancellous bone structures based on noninvasive high-resolution CT measurements are representative for trabecular microstructures assessed from histologic bone sections. The study demonstrates the potential of high-resolution CT imaging for in vivo applications of quantitative bone morphometry. This is especially true for repetitive follow-up measurements, which cannot be performed using histologic sections. Additionally, the method offers an easy access to the three-dimensional structure of trabecular bone, which is mandatory for the analyses of the anisotropic mechanical behavior of cancellous bone.

Morphological grades of regression in osteosarcoma after polychemotherapy — Study COSS 80

SummaryThe histologic grade of regression of 50 osteosarcomas after polychemotherapy — according to the protocol study, COSS 80 — was classified on a six-stage regression scale; 56% of all patients responded well to chemotherapy regression grades I, II, and III and no significant difference between BCD- and CPL-treated patients could be found. Tumors under 10 cm in length responded better to chemotherapy than those of greater length and there was a good correlation between the clinical estimation of tumor regression and progression and the histologic grade of regression.

3D computed X-ray tomography of human cancellous bone at 8 μm spatial and 10−4 energy resolution

Human cancellous bone was imaged and its absorptive density accurately measured in three dimensions (3D), nondestructively and at high spatial resolution by means of computerized microtomography (microCT). Essential for achieving the resolution and accuracy was the use of monoenergetic synchrotron radiation (SR) which avoided beam hardening effects, secured excellent contrast conditions including the option of energy-modulated contrast, and yet provided high intensity. To verify the resolution, we selected objects of approximately 8 micron size that could be observed on tomograms and correlated them in a unique manner to their counter images seen in histological sections prepared from the same specimen volume. Thus we have shown that the resolution expected from the voxel size of 8 microns used in the microCT process is in effect also attained in our results. In achieving the present results no X-ray-optical magnification was used. From microCT studies of composites (Bonse et al., X-ray tomographic microscopy (XTM) applied to carbon-fibre composites. In: Materlik G, ed. HASYLAB Jahresbericht 1990. Hamburg: DESY, 1990; 567-568) we know that by including X-ray magnification a resolution below 2 microns is obtained. Therefore, with foreseeable development of our microCT method, the 3D and nondestructive investigation of structures in mineralized bone on the 2 micron level is feasible. For example, it should be possible to study tomographically the 3D distribution and amount of osteoclastic resorption in the surrounding bone structure.

The nature of giant cell tumor of bone

Abstract Giant cell tumor of bone (GCT) is a locally osteolytic tumor with variable aggressiveness. In rare cases, pulmonary metastasis can be observed. The lesion most frequently occurs in the epiphysis of long tubular bones of the knee region, predominantly affecting young adults after closure of the growth plate. The characteristic histological appearance of GCT displays a high number of osteoclast-like multinucleated giant cells, which resulted in the classification “osteoclastoma” or “giant cell tumor”. Apart from the multinucleated giant cells, there are two mononuclear cell types in GCT. The first one has a round morphology and resembles monocytes. The second cell type is the spindle-shaped, fibroblast-like stromal cell. Cell culture experiments with GCT cells revealed the stromal cell to be the proliferating component of the GCT. The other two cell types, the monocyte and the multinucleated giant cell, were lost after a few cell culture passages. Furthermore, latest results from GCT reveal that the stromal cells secrete a variety of cytokines and differentiation factors, including MCP1, ODF, and M-CSF. These molecules are monocyte chemoattractants and are essential for osteoclast differentiation, suggesting that the stromal cell stimulates blood monocyte immigration into tumor tissue and enhances their fusion into osteoclast-like, multinucleated giant cells. The multinucleated giant cell itself resembles a normal osteoclast that is able to resorb bone leading to extended osteolysis. This new model of GCT genesis supports the hypothesis that the stromal cell is the neoplastic component whilst the monocytes and the multinucleated giant cells are just reactive components of this tumor. Taking this into consideration, the nomenclature of the “giant cell tumor” needs to be reconsidered.

Corroded Nitinol Wires in Explanted Aortic Endografts: An Important Mechanism of Failure?

Purpose: To analyze surface alterations and fractures observed in the nitinol stent wires of explanted endovascular grafts used for treatment of abdominal aortic aneurysm. Methods: Twenty-one explanted Stentor devices and 1 Cragg stent were received from investigators in Germany and France. After macroscopy and photography, the explants were cleaned and the polyester coating removed. The frame was examined completely by stereomicroscopy, and irregularities were assayed by scanning electron microscopy and energy dispersive x-ray analysis (EDAX). The observed alterations were classified according to stereomicroscopic and electron microscopic morphology. Results: The mean implantation interval for the endografts was 29.1 ± 13.2 months (range 5–46). All examined explants, even those retrieved after only a few months in situ, showed pitlike surface damage 10–25 µm in diameter. Larger, irregularly shaped surface alterations were observed in ∼70% of the explants. Older explants (age >32 months) presented vast regions of decay, with bending of the wire and stress cracks in some areas. EDAX examination revealed decreased nickel concentration in the corroded regions. Conclusions: Corrosion of the nitinol wire in endovascular grafts is confirmed. Presumably, the observed pitting and irregularly shaped corrosion defects are the precursors of material failure. They weaken the thin wire, which leads to stress cracks and eventually fracture of the stent wire under circulatory pulsation. Cell-induced electrochemical corrosion and active cellular destruction of surfaces are well-known mechanisms that must be investigated for their possible roles in the corrosion of stent metals.

The thickness of human vertebral cortical bone and its changes in aging and osteoporosis : A histomorphometric analysis of the complete spinal column from thirty-seven autopsy specimens

The object of this study was to analyze the cortical thickness (Ct.Th) of the ventral and dorsal shell of the vertebral bodies throughout the human spine in aging and in osteoporosis. Therefore, the complete front column of the spine of 26 autopsy cases (aged 17–90, mean 42 years) without diseases affecting the skeleton and of 11 cases (aged 58–92, mean 77 years) with proven osteoporosis were removed. A sagittal segment prepared through the center of all vertebral bodies was undecalcified, embedded in plastic, ground to a 1 mm thick block, and stained using a modification of the von Kossa method. The analysis included the measurement of the mean cortical thickness of both the ventral and dorsal shell, respectively (from the third cervical to the fifth lumbar vertebral body). The qualitative investigation of the structure of the cortical ring completed the analysis. The presented data revealed a biphasic curve for both the ventral and dorsal shell, skeletally intact with high values of the cortical thickness in the cervical spine (285 μm), and a decrease in the thoracic (244 μm) and an increase in the lumbar spine (290 μm). The mean thickness of the ventral shell is in general greater than the thickness of the dorsal shell in both skeletally normal and osteoporotic cases. The cortical thickness of the spine showed no gender‐specific differences (p = NS). There was a slight decrease of the cortical thickness with aging; however, this decrease and the correlation of cortical thickness to age was only significant below vertebral body T8 (r = 0.225–0.574; pr < 0.05–0.005). Most interestingly, however, osteoporosis presents itself with a highly significant loss of cortical thickness throughout the whole spine. This decrease of cortical thickness was more marked in the dorsal shell (p < 0.05) than in the ventral shell (ventral from C3 to T6 [p < 0.05] below T6 [p = NS]). We therefore conclude that in osteoporosis the loss of spinal bone mass is not only a loss of trabecular structure but also a loss of cortical thickness. Furthermore, these results may explain the development of regions of least resistance within the spine in aging and the clustering of osteoporotic fractures in the lower thoracic and lumbar spine.

Undecalcified preparation of bone tissue: Report of technical experience and development of new methods

For reliable quantitative and qualitative analysis of bone specimens undecalcified preparation is essential. The “conventional” technique for this purpose is embedding in methylmethacrylate. Larger bone specimens, highly sclerotic specimens, cortical bone or bone implants consisting of metals or ceramics require modifications of this technique or completely new methods. We report our experience with the undecalcified preparation of 47700 bone specimens. New techniques such as the cutting of large area sections up to a size of 5×6 cm and grinding procedures for completely artefact-free preparation which are applied in special cases are also described. A new technique of combinded two- and three-dimensional analysis of bone specimens is presented. In our experience these methods are fundamental for morphological investigation of bone.

Increase of vertebral density by combination therapy with pulsatile 1-38hPTH and sequential addition of calcitonin nasal spray in osteoporotic patients

SummaryCombination therapy with the biologically active (1–38) human parathyroid hormone peptide and calcitonin using pulsatile and sequential activation of the skeleton for 14 months in patients with low-turnover osteoporosis resulted in an increase in trabecular bone mass. These favorable responses were observed without any significant changes in cortical (forearm) bone mass content.