F. Melsen

An Endocytic Pathway Essential for Renal Uptake and Activation of the Steroid 25-(OH) Vitamin D3

Steroid hormones may enter cells by diffusion through the plasma membrane. However, we demonstrate here that some steroid hormones are taken up by receptor-mediated endocytosis of steroid-carrier complexes. We show that 25-(OH) vitamin D3 in complex with its plasma carrier, the vitamin D-binding protein, is filtered through the glomerulus and reabsorbed in the proximal tubules by the endocytic receptor megalin. Endocytosis is required to preserve 25-(OH) vitamin D3 and to deliver to the cells the precursor for generation of 1,25-(OH)2 vitamin D3, a regulator of the calcium metabolism. Megalin-/- mice are unable to retrieve the steroid from the glomerular filtrate and develop vitamin D deficiency and bone disease.

Cancellous Bone Remodeling Occurs in Specialized Compartments Lined by Cells Expressing Osteoblastic Markers

We describe a sinus, referred to as a bone remodeling compartment (BRC), which is intimately associated with cancellous bone remodeling. The compartment is lined on its marrow side by flattened cells and on its osseous side by the remodeling bone surface, resembling a roof of flattened cells covering the bone surface. The flat marrow lining cells are in continuity with the bone lining cells at the margins of the BRC. We examined a large number of diagnostic bone biopsy specimens received during recent years in the department. Furthermore, 10 patients (8 women and 2 men, median age 56 [40–69] years) with the high turnover disease of primary hyperparathyroidism who were treated with parathyroidectomy and followed for 3 years were included in the histomorphometric study. Bone samples for the immuno‐enzyme staining were obtained from an amputated extremity of child. The total cancellous bone surface covered by BRC decreases by 50% (p < 0.05) following normalization of turnover and is paralleled by a similar 50% decrease in remodeling surface (p < 0.05). The entire eroded surface and two‐thirds of the osteoid surface are covered by a BRC. BRC‐covered uncompleted walls are 30% (p < 0.05) thinner than those without a BRC. This indicates that the BRC is invariably associated with the early phases of bone remodeling, that is, bone resorption, whereas it closes during the late part of bone formation. Immuno‐enzyme staining shows that the flat marrow lining cells are positive for alkaline phosphatase, osteocalcin, and osteonectin, suggesting that they are bone cells. The first step in cancellous bone remodeling is thought to be the lining cells digesting the unmineralized matrix membrane followed by their disappearance and the arrival of the bone multicellular unit (BMU). We suggest that the lining cell barrier persists during bone remodeling; that the old lining cells become the marrow lining cells, allowing bone resorption and bone formation to proceed under a common roof of lining cells; that, at the end of bone formation, new bone lining cells derived from the flattened osteoblasts replace the marrow lining cells thereby closing the BRC; and that the two layers of lining cells eventually becomes a single layer. The integrity of the osteocyte‐lining cell system is reestablished by the new generation of lining cells. The BRC most likely serves multiple purposes, including efficient exchange of matrix constituents and minerals, routing, monitoring, or modulating bone cell recruitment, and possibly the anatomical basis for the coupling of bone remodeling.

Biologically meaningful determinants of the in vitro strength of lumbar vertebrae

Applying unbiased stereological methods and a new stereological parameter, star volume of cancellous bone, the bone structure of the first vertebral body was examined and compared with the compressive strength of the second lumbar vertebra. The material came from eight males, aged 33-69 years (mean 49 years) and seven women, aged 22-87 years (mean 52 years) without malignant or metabolic bone disease. From these individuals, first and second lumbar vertebral body were obtained at autopsy. The heights and weights of the individuals were recorded. The following structural parameters were estimated on undecalcified, seven-microns, Goldner-Trichrome stained vertical sections: fractional volume of trabecular bone (BV/TV%), mean trabecular thickness (Tb.Th.l1 microns), trabecular star volume (V*tr mm3), marrow space star volume (V* m.space mm3), and mean thickness of the lateral cortical ring (microns). The compressive strength of whole vertebral body, mean cross sectional area (cm2), and ash density (g/cm3) were estimated and the data were compared to bone histomorphometric estimates. A significant decrease with age for all parameters was found except for marrow space star volume, which increased. With compressive strength as the dependent variable and all other parameters as independent variables, it was shown by standard multiple regression analysis that the in vitro tested compressive strength could be predicted from mean cortical thickness, mean cross sectional area, and marrow space star volume or ash density with a multiple, squared coefficient of regression (r2) of 0.95 when the height and sex of the individual were known.(ABSTRACT TRUNCATED AT 250 WORDS)

A randomized study on the effects of estrogen/gestagen or high dose oral calcium on trabecular bone remodeling in postmenopausal osteoporosis.

Thirty-seven patients with postmenopausal crush fracture osteoporosis were randomized to oral cyclic estrogen/gestagen (n = 20) or oral calcium (2000 mg elemental calcium per day) (n = 17). Fourteen in each group completed 1 year of treatment. Iliac crest bone biopsies were obtained after intravital double labeling with tetracycline before and after treatment in 10 patients on estrogen/gestagen and 11 patients on calcium. In the estrogen/gestagen group the activation frequency in trabecular bone decreased from 0.52 + 0.11 (SEM) year−1 to 0.27 + 0.08 year−1 (p < 0.01). No significant changes were found in resorption or formation periods. The osteoid surfaces and the mineralizing surfaces decreased (p < 0.05), whereas the decrease in eroded surfaces was insignificant. Furthermore, no significant changes were observed in final resorption depth, wall thickness or bone balance per remodeling cycle. Serum alkaline phosphatase and renal hydroxyproline excretion decreased during treatment (p < 0.002), whereas the lumbar bone mineral content (BMC) increased {ifp < 0.01}. In the calcium group the extent and thickness of osteoid surfaces decreased (p < 0.05) without significant changes in activation frequency. Serum alkaline phosphatase and renal hydroxyproline excretion decreased during treatment (p < 0.02). No significant changes were observed in lumbar BMC or the other histomorphometric parameters. The study supports that the positive effect of estrogen/gestagen on BMC can be explained by a reduction in the activation frequency of new remodeling cycles leading to a decreased remodeling space and an increase in mean bone age. There is no evidence of a positive balance per remodeling cycle. High dose calcium may improve osteoid mineralization, but there is no histomorphometric evidence of a significant reduction in activation frequency or a more positive balance per remodeling cycle.

Age- and sex-related changes in iliac cortical bone mass and remodeling

Iliac crest bone biopsies were obtained from 64 normal individuals (41 women and 23 men) aged 19-90 (mean 48.2) years. Thirty-four were double-labeled with tetracycline before biopsy. The following variables were measured in all biopsies: biopsy core width (C.Wi), absolute (A.Ct.Wi) and fractional (F.Ct.Wi) cortical width, absolute (A.Cn.Wi) and fractional (F.Cn.Wi) cancellous width, cortical porosity (Ct.Po), osteon diameter (On.Dm), Haversian canal diameter (Ha.Ca.Dm), and wall thickness (W.Th). In the tetracycline-labeled biopsies the typical cortical remodeling cycle was reconstructed and the activation frequency was estimated. A negative cortical bone balance with aging was found in both sexes. In females F.Ct.Wi decreased (p < 0.02) with aging because of marrow expansion (p < 0.05). Furthermore, Ct.Po increased (p < 0.001) because of a decrease in W.Th (p < 0.01) and an increase in H.Ca.Dm (p < 0.001). In males the negative cortical bone balance with aging was exclusively caused by an increase in Ct.Po (p < 0.001) partially explained by an expanding H.Ca.Dm (p < 0.01). The On.Dm increased with aging (p < 0.01), but surprisingly, no fall in W.Th was observed. Reconstruction of the remodeling cycle did not reveal any significant difference between younger women and men. However, the activation frequency rose from 0.5 per year in premenopausal to 1.0 per year in postmenopausal women (p < 0.001), giving rise to a high turnover state, an increase in the remodeling space, and thereby the porosity in the cortical bone immediately after menopause. The present study has shown a reduction in cortical bone mass in elderly people, compared with younger, which may be explained by an age-related remodeling imbalance. This reduction is further increased in women in the postmenopausal state because of a postmenopausal accelerated bone turnover.

Star volume of marrow space and trabeculae of the first lumbar vertebra: Sampling efficiency and biological variation

The decrease in the amount of trabecular bone which is seen with age cannot solely be explained by thinning of trabeculae but must also be due to a loss of structural trabecular bone leading to a discontinuity in the trabecular network. Due to the complex architecture and the anisotropy of bone it is difficult to demonstrate this structural change by conventional histomorphometry. Unbiased stereological estimators can however be obtained from anisotropic structures when using vertical sections and a specially designed anisotropic test system. This combined with a new and unbiased stereological parameter for bone structure the star volume can be of major importance in clarifying histological changes of bone structure. The star volume is defined as the mean volume of all the parts of an object which can be seen unobscured in all directions from a particular point with the mean value taken over all points inside the object. It is defined for any type of objects including cavities like marrow space and networks like the trabecular system. Measurements are performed using a frame and a grid with points and lines. The material investigated was the first lumbar vertebra obtained from two females and six males with ages 26 to 75 years without malignant or metabolic bone diseases. The sampling procedure was as required for vertical sections. Results did show a highly significant, five-fold increase in the star volume of the marrow space with age; no such age correlation was found for the star volume of the trabeculae. The only explanation for such an increase in the size of the marrow space is by removing or perforating trabecular bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Trabecular bone remodeling and bone balance in hyperthyroidism

In vivo tetracycline double-labeled iliac crest bone biopsies from 15 hyperthyroid patients were used for the reconstruction of curves describing the variation of resorption depth and formation thickness with time. The curves emerging were compared to curves reconstructed from 13 age- and sex-matched normal individuals (mean age 44 years). The median function period for resorptive cells in hyperthyroid patients (16 days) was about one-third the resorptive period in normals (51 days). No significant difference between the osteoclast-, mononuclear-, or preosteoblast-like cell resorption depths could be demonstrated between the two groups. Consequently, the median resorption rate in hyperthyroid patients (3.8 microns/day) was more than 3 times higher than the value in the control group (1.1 micron/day). Median Sigma, was shorter in the hyperthyroid group (109 days) than in the control group (151 days, P less than 0.05), as was the median initial mineralization lag time (5 and 16 days, respectively, P less than 0.01). No significant difference between the measured mean completed wall thickness (mcwT) values in the hyperthyroid groups and the control group could be demonstrated (58.1 and 60.5 micron respectively). Median initial mineralization rate in the hyperthyroid group (1.2 micron3/micron2 per day) was not significantly higher than the value calculated in the control group (0.9 micron3/micron2 per day), but median initial matrix appositional rate in hyperthyroid (4.8 microns3/micron2 per day) was 3 times higher than the value calculated for normals (1.6 micron3/micron2 per day) (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect

Megalin is an endocytic receptor highly expressed in the proximal tubules of the kidney. Recently, we demonstrated that this receptor is essential for the renal uptake and conversion of 25‐OH vitamin D3 to 1,25‐(OH)2 vitamin D3, a central step in vitamin D and bone metabolism. Unfortunately, the perinatal lethality of the conventional megalin knockout mouse model precluded the detailed analysis of the significance of megalin for calcium homeostasis and bone turnover in vivo. Here, we have generated a new mouse model with conditional inactivation of the megalin gene in the kidney by using Cre recombinase. Animals with a renal‐specific receptor gene defect were viable and fertile. However, lack of receptor expression in the kidney results in plasma vitamin D deficiency, in hypocalcemia and in severe bone disease, characterized by a decrease in bone mineral content, an increase in osteoid surfaces, and a lack of mineralizing activity. These features are consistent with osteomalacia (softening of the bones) as a consequence of hypovitaminosis D and demonstrate the crucial importance of the megalin pathway for systemic calcium homeostasis and bone metabolism.

Primary hyperparathyroidism: Iliac crest trabecular bone volume, structure, remodeling, and balance evaluated by histomorphometric methods

Iliac bone biopsies from 69 patients (48 females, 21 males; median age 58 years; range 17-79 years) with primary hyperparathyroidism (PHP) were examined, and static histomorphometric parameters compared to 30 age- and sex-matched normal controls. The control group for the dynamic parameters constituted 20 sex-matched younger normal controls. Fractional volume of trabecular bone was normal, but the trabeculae were thinner (p less than 0.05) in PHP. The structural parameters marrow space star volume, intertrabecular distance, and mean trabecular plate density were not significantly different in PHP patients compared to normal controls, but the age-related increase, for females, in marrow space star volume and decrease, for both sexes, in mean plate density observed in the controls were not noticed in the PHP group. Trabecular bone remodeling was found significantly increased in the PHP patients reflected by increased extension of eroded (p less than 0.001), osteoid (p less than 0.001), and labeled surfaces (p less than 0.05). The activation frequency was increased by approximately 50% (p less than 0.001). Neither PHP patients nor controls showed age-related decrease in trabecular thickness, and accordingly in both groups the bone balance per remodeling cycle was very close to and not significantly different from zero. Normal postmenopausal women (age greater than or equal to 50 yr) had lower trabecular bone volume (p less than 0.001) and higher intertrabecular distance than normal pre-menopausal women (age less than 50 yr).(ABSTRACT TRUNCATED AT 250 WORDS)

Trabecular bone remodeling and balance in primary hyperparathyroidism

The total remodeling sequences in 19 primary hyperparathyroid patients and 16 approximately age-matched and sex-matched controls were reconstructed from histomorphometric analyses of bone specimens obtained after intravital tetracycline double labeling. In the primary hyperparathyroid group the total amount of work performed by resorptive cells was reduced, as indicated by the significantly lower three-dimensional mononuclear and preosteoblast-like cell resorption depths (35.8 microns vs 44.5 microns in normals, P less than 0.01 and 45.3 microns vs 56.6 microns in normals, P less than 0.01, respectively). The active resorption period (i.e., the function period for osteoclasts and mononuclear cells) was reduced to 19 days compared to 29 days in normals (P less than 0.05), but no difference with respect to bone resorption rates could be demonstrated between the two groups. The median bone formation period (Sigmaf) in primary hyperparathyroid patients was not different from the value obtained in normals (172 days vs 134 days, respectively), and the matrix appositional rate (Ama), as well as the mineralization lag time (tm), were also unchanged. The initial mineralization rate (Ami(i)) was not significantly different from the value obtained in normals, but averaged over the total bone formation period, a reduced mineralization rate could be demonstrated (0.32 micron3/micron2 per day vs 0.46 micron3/micron2 per day in normals, P less than 0.01). The measured final three-dimensional thickness of bone formed during Sigmaf (mcwTm) was reduced in the primary hyperparathyroid group (51.1 microns vs 55.9 microns in normals, P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)