Hormonal regulation of calcium homeostasis in two breeds of dogs during growth at different rates.

Growing giant-breed dogs are more susceptible to developing skeletal disorders than small-breed dogs when raised on diets with deficient or excessive Ca content. Differential hormonal regulation of Ca homeostasis in dogs with different growth rates was investigated in Great Danes (GD, n = 9) and Miniature Poodles (MP, n = 8). All animals were raised on the same balanced diet and under identical conditions. Calciotropic and growth-regulating hormones were measured. Production and clearance of 1,25-dihydroxycholecalciferol (1,25[OH]2D3) were investigated with the aid of [3H]-1,25(OH)2D3 and renal messenger RNA abundance of 1 alpha-hydroxylase and 24-hydroxylase. Intestinal, renal, and skeletal Ca handling were evaluated with the aid of 45Ca balance studies. Skeletal development was evaluated by radiology and histomorphometry. Great Danes had greater (P < 0.001) growth rates than MP, as indicated by the 17-fold greater body weight gain, by increased longitudinal growth reflected in the increased (P < 0.05) gain in length of the radius and ulna, and by increased (P < 0.001) growth plate thickness. These findings were accompanied in GD by greater (P < 0.05) plasma GH and IGF-I concentrations. Effects were observed for vitamin D3 metabolism, such as greater (P < 0.01) plasma 1,25(OH)2D3 concentrations due to decreased (P < 0.01) clearance rather than increased production of 1,25(OH)2D3, and decreased (P < 0.01) plasma 24,25-dihydroxycholecalciferol (24,25[OH]2D3) concentrations likely due to competitive inhibition of the production of 24,25(OH)2D3. These findings were accompanied in both breeds by a limited hormonal regulation of Ca and P absorption at the intestinal level, and in GD by increased (P < 0.05) renal reabsorption of inorganic P (Pi) compared with MP, resulting in greater (P < 0.01) Pi retention and greater (P < 0.01) plasma Pi concentrations. Bone turnover, resorption, and formation were greater (P < 0.01) in GD than in MP. In addition, GD had more irregular (P < 0.01) growth plates than MP, accompanied by disorders of endochondral ossification. It is suggested that in GD, increased calcitonin levels and/or a relative deficiency in 24,25(OH)2D3 at the growth-plate level may both be responsible for the retarded maturation of chondrocytes, resulting in retained cartilage cones and osteochondrosis, and this may be a pathophysiological factor for the increased susceptibility of large breed dogs to developing skeletal disorders.

Trabecular bone's mechanical properties are affected by its non-uniform mineral distribution.

The bone remodeling process takes place at the surface of trabeculae and results in a non-uniform mineral distribution. This will affect the mechanical properties of cancellous bone, because the properties of bone tissue depend on its mineral content. We investigated how large this effect is by simulating several non-uniform mineral distributions in 3D finite element models of human trabecular bone and calculating the apparent stiffness of these models. In the 'linear model' we assumed a linear relation between mineral content and Young's modulus of the tissue. In the 'exponential model' we included an empirical exponential relation in the model. When the linear model was used the mineral distribution slightly changed the apparent stiffness, the difference varied between an 8% decrease and a 4% increase compared to the uniform model with the same BMD. The exponential model resulted in up to 20% increased apparent stiffness in the main load-bearing direction. A thin less mineralized surface layer (28 microm) and highly mineralized interstitial bone (mimicking mineralization resulting from anti-resorptive treatment) resulted in the highest stiffness. This could explain large reductions in fracture risk resulting from small increases in BMD. The non-uniform mineral distribution could also explain why bone tissue stiffness determined using nano-indentation is usually higher than finite element (FE)-determined stiffness. We conclude that the non-uniform mineral distribution in trabeculae does affect the mechanical properties of cancellous bone and that the tissue stiffness determined using FE-modeling could be improved by including detailed information about mineral distribution in trabeculae in the models.

Immunolocalization and quantification of noncollagenous bone matrix proteins in methylmethacrylate-embedded adult human bone in combination with histomorphometry.

The noncollagenous proteins (NCPs) in the bone matrix comprise growth factors with distinct cellular effects and a series of proteins with less clear biological actions. In order to understand the role of these proteins in bone metabolism and in bone diseases, it is crucial to determine their localization and quantity in normal and pathological bone. We have developed an immunohistochemical method to detect osteopontin, osteocalcin, bone sialoprotein, osteonectin, decorin, biglycan, and the growth factors transforming growth factor-beta, insulin-like growth factor-I, and bone morphogenetic protein-2 both in bone matrix and in bone cells of adult human bone embedded in methylmethacrylate. Immunohistochemistry and standard bone histomorphometry in adjacent sections allows the localization of the proteins to metabolically active sites in bone. The protocol works with several fixatives and with bone specimens obtained and embedded to over 20 years ago. Most importantly, we developed a procedure to specifically stain the mineralized matrix green in combination with a red staining of the NCPs. Using digital image analysis it is possible to quantify the relative amounts of NCPs (microm2 NCP area/microm2 mineralized matrix area). Within one biopsy of normal bone cut at four different heights (at a distance of 100 microm), two adjacent sections were stained either for osteopontin or osteonectin. Thirty trabecular and 20 cortical microscopic fields were measured, and the NCP:mineralized matrix ratio was calculated. Stepwise analysis of the standard error of the mean of the NCP:mineralized matrix ratios showed that measuring about 50 microscopic fields is sufficient to obtain representative data with a small confidence interval. In conclusion, the present procedure enables to quantify NCPs and to relate their presence to metabolically active sites in bone. The quantification provides the opportunity to monitor differences in distribution (e.g., cortical vs. trabecular) and differences between normal and pathological conditions and to assess changes in matrix composition during treatment. This can be done by reanalyzing bone biopsies obtained in the past, e.g., during clinical trials. Therefore, the present technique will be a valuable tool for the study of noncollagenous bone matrix proteins in human bone.

A thionin stain for visualizing bone cells, mineralizing fronts and cement lines in undecalcified bone sections.

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored in the dark at 7 C.

Effects of 24R,25-dihydroxyvitamin D3 in combination with 1 alpha-hydroxyvitamin D3 in predialysis renal insufficiency: biochemistry and histomorphometry of cancellous bone.

The effect of combined administration of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and 1 alpha-hydroxyvitamin D3 (1 alpha-(OH)D3) was studied in 24 non-dialyzed patients with chronic renal insufficiency (CRI), matched pairwise as to age, sex, and creatinine clearance (Cr.cl). Low Ca intake had been supplemented beforehand. Then, 1 alpha-(OH)D3 (mean dose 0.55 micrograms daily) was given orally to all patients for 3 months (T0 to T3). Subsequently, patients were assigned randomly to 6 months further treatment either with 1 alpha-(OH)D3 alone (Group A) or with 1 alpha-(OH)D3 plus a high dosage of 24,25-(OH)2D3 (50 micrograms orally, twice weekly) (Group B). Histomorphometry was performed at T0, T3, and T9. In both groups iPTH was equally suppressed, into the lower normal range. Whereas in Group A, serum Ca rose steadily and Cr.cl declined, in Group B both parameters levelled off between T6 and T9. At T9, in Group A the elevated resorption and osteoid indices had normalized markedly, but osteoblasts (Ob.Pm) and mineralizing boundaries (M.Bd) were depressed considerably between T3 and T9. In contrast, in Group B, preservation of Ob.Pm and improved mineralizing activity were observed (M.Bd at T9 > T3 > T0). Resorption indices hardly changed. In the patients with high Ob.Pm at T0, cancellous bone area increased significantly. This was not observed in Group A. Thus, in Group B, osteoblast recruitment appeared maintained and M.Bd appeared normalized. Decline of remodeling toward an adynamic state with an increased risk of hypercalcemia appeared prevented.

Course of bone mass during and after hormonal replacement therapy with and without addition of nandrolone decanoate.

A total of 33 women with postmenopausal osteoporosis were matched pairwise by age, years since menopause, and body mass index and randomized to receive either cyclic estrogen-progestagen replacement treatment (group 1) or the same treatment plus nandrolone decanoate (ND; group 2). Both groups were treated during 3 years and subsequently followed for another year off treatment. A year after cessation of the treatment the distal forearm bone mineral content in group 2 was significantly higher than that in group 1. Bone mass measurements in the axial skeleton already showed a significant difference in favor of group 2 after 3 years treatment, which persisted during the year off treatment. The decline in lumbar bone mineral mass and density in the 1 year off treatment was similar in both groups. Correction for body mass did not change these results. Bone turnover parameters did not show significant differences between the two groups after cessation of treatment. A higher muscle mass, induced by ND, could partly explain the differences between the groups since even 1 year after treatment was stopped an increased serum creatinine level was still observed in group 2.

Changes of interstitial bone thickness with age in men and women.

Mean interstitial bone thickness has been estimated from mean trabecular thickness and mean wall thickness (It.Th = Tb.Th-2*W.Th) with varying results. Some authors found age-related changes of It.Th while others did not. We measured It. Width (It.Wi, 2D) directly, in cancellous iliac crest bone from 23 women aged 20-78 yrs. and 21 men aged 23-74 yrs. At grid-selected sites coupled measurements were done of Tb.Wi and It.Wi, together with W.Wi. It.Wi was defined as the distance between cement lines nearest to the surface at both sides of a trabecula. A thionine stain was used to visualize the cement lines. When the persons studied were divided into two age groups: 20-49 yrs (Group 1) and 50-79 yrs (Group 2) significant but opposite age-related trends were seen both in men and women. Group 1: Men: r = -.68; p < .02; n = 11; Women: r = -.68; p < .02; n = 11. Group 2: Men: r = .79; p < .01; n = 10. Women: r = .59; p < .05; n = 12 (r = coeff. of correlation). Men: It.Wi 63.3 microns (3rd decade [dec.]); 50.8 microns (6th dec.); 67.8 microns (8th dec.). Women: It.Wi 63.7 microns (3rd dec.); 48.3 microns (6th dec.); 63.8 microns (8th dec.). Measured It.Wi values appeared considerably larger than their calculated counterparts (mean delta: 35%). The decline of It.Wi is interpreted as a sign of negative bone balance at the BMU level, leading to thinning of trabeculae, the subsequent increase has to be the result of both declining resorption depth and disappearance of the thinner trabeculae.(ABSTRACT TRUNCATED AT 250 WORDS)

Can nandrolone add to the effect of hormonal replacement therapy in postmenopausal osteoporosis?

Thirty-six women with postmenopausal osteoporosis (31 of them with at least one non-traumatic vertebral compression fracture) were matched pair-wise as to age, years since menopause and body mass index and randomized to receive either cyclical estrogen-progestagen replacement treatment (group 1) or the same treatment plus nandrolone decanoate (group 2). During the first year of treatment in both groups the forearm BMC (SPA) rose proximally and distally 2-3%. Over 2 years the increments of forearm BMC in both groups were up to 4.5%. Lumbar BMC (DPA) rose in both groups nearly 10% over the first year and 12-12.5% over 2 years. The cancellous bone density of L3 (QCT) showed in 6 months an increase of 21% in group 1 and 29% in group 2 to subsequently stay at that level. All these changes from the basal levels were highly significant but there were no significant differences between the two groups. These two conclusions were also drawn with regard to the induced fall of serum alkaline phosphatase (-23%), osteocalcin (-35% to -44%) and procollagen I (-15% to -22%) and of the fasting urinary hydroxyproline (-33% to -36%). No significant increase in the number of newly deformed vertebrae occurred in 2 years.

Noninvasive axial and peripheral assessment of bone mineral content: a comparison between osteoporotic women and normal subjects.

We compared different methods of bone densitometry in women with spinal osteoporosis and normal subjects to assess their discriminatory capability. The methods used included: quantitative computed tomography of the spine (QCT) specified as to trabecular (QCTtrab) and cortical bone (QCTcort), dual-photon absorptiometry of the spine (DPAspine), single-photon absorptiometry of the distal and proximal forearm (SPAdist and SPAprox), and quantitative roentgen microdensitometry of the phalanx (QMD). A total of 25 postmenopausal osteoporotic women and 24 healthy comparison subjects matched for age and years since menopause were studied. In the osteoporotic group an average decrement of the axial bone mineral density of -50% (p less than 0.001) and -20% (p less than 0.001) were observed for QCTtrab and QCTcort, respectively. For DPAspine, SPAdist, SPAprox, and QMD the difference between normal and osteoporotic subjects was -20% (p less than 0.001), -12% (p less than 0.05), -7% (NS), and -6% (NS), respectively. With the peripheral measurements (SPA and QMD), alone or in combination, no adequate discrimination between women with or without vertebral compression fractures could be obtained. Although QCTtrab showed the highest diagnostic sensitivity (81%), it appears not to be superior to DPAspine. Combinations of the various axial and peripheral measurements did not result in an essentially better sensitivity. In normal women as well as in osteoporotic individuals the trabecular and cortical QCT measurements showed two opposite trends, suggesting an increase in cortical and a decrease in trabecular density from L1 to L3.

Effects of 1 alpha-hydroxyvitamin D3 on various stages of predialysis renal bone disease.

Biochemical parameters of mineral metabolism and bone histomorphometric measurements--both static and dynamic--were studied in 27 to 29 patients with chronic renal failure before (T0) and after 3 months treatment (T3) with 1 alpha-hydroxyvitamin D3 (1 alpha (OH)D3; average daily dose 0.55 micrograms). In none of the biopsies was a positive aluminum stain found. Fourteen patients had an osteoblast seams length (Ob.Pm) of less than 4% (Group I) and high osteoid parameters, whereas 13 patients (Group II, Ob.Pm greater than 4%) also had clear histological signs of hyperparathyroidism. Group II had lower creatinine clearance and serum calcium, but higher iPTH values. Treatment with 1 alpha (OH)D3 resulted in a substantial suppression of secondary hyperparathyroidism in Group II, with a fall in Ob.Pm, the cancellous bone perimeter occupied by tetracycline double label and osteoclast perimeter (Oc.Pm). In Group II treatment resulted in the development of a positive correlation between Ob.Pm and the number of osteoclasts (N.Oc). With treatment the (thionine) mineralization front rose in both groups, but osteoid seams length did not fall. When calculated for both groups together, before and after treatment serum calcium was negatively correlated with osteoid seams length, while a positive correlation was found with the mineralization front. This study provides an indication that, in progressive renal bone disease in which aluminum intoxication has been excluded, hyperosteoidosis precedes the development of secondary hyperparathyroidism. Furthermore, the study shows that treatment with 1 alpha (OH)D3 suppresses secondary hyperparathyroidism and results in a moderate increase of mineralization.

Age-related changes in cancellous bone structure. A two-dimensional study in the transiliac and iliac crest biopsy sites.

In 94 pairs of large sections of normal iliac bone (53 men, 41 women, aged 20-80 years) we confirmed trabecular bone area (B.Ar) and the mean number of trabeculae per mm (Tb.N) to decline with age, in areas representative of the transiliac (TI) and iliac crest (IC) biopsy in both sexes, whereas mean trabecular width (Tb.Wi) did not decrease. Our data also confirm that disappearance of entire elements is the main event in age-related loss of trabecular bone. In addition, our study demonstrated that Tb.Wi is lower in women than in men in the IC site, and a preferential loss of the thinner trabeculae could be seen in this group. Up to 50 years, Tb.N was found higher in women in both sites. B.Ar and Tb.Wi values are markedly higher in the TI site (+/- 15%); Tb.N values were similar. Measured and calculated Tb.Wi values show systematic differences. In a pilot study in 14 IC biopsies with widely diverging B.Ar values, we tested the validity of several equations currently used for derived parameters in the structural analysis of cancellous bone sections. The equation for mean trabecular number passed the test, whereas the validity of the equations for mean trabecular separation and Tb.Wi appeared limited. We found anisotropy of iliac bone to increase as B.Ar decreases.

Bone appositional rate and percentage of doubly and singly labeled surfaces: comparison of data from 5 and 20 microns sections.

On theoretical grounds, it seemed plausible that the apparent distance between two fluorescent labels can surpass the actual distance between them by a percentage that is related to the true distance: section thickness ratio. For similar reasons, the percentage of double labels discernible as such would be influenced by the same ratio. These assumptions were tested in a series of doubly labeled biopsies in which a large variation of appositional rates had been found. The apparent mean distance between labels and, hence, the appositional rate as measured in 20 microns sections was significantly lower than in the 5 microns sections from the same biopsies. Yet, 20 microns sections cannot be used to counteract distortion by oblique sectioning because of the bias obscuring the lower range of true interlabel distances. The percentage of doubly labeled surfaces was from 13 to over 50% lower in the 20 microns sections. Thus, overlap of double labels by geometrical projection in 20 microns sections causes a considerable reduction of formation rates derived from the parameters investigated. The problem is resolved, to a large extent, by using sections as thin as possible.

Software for bone histomorphometry by means of a digitizer.

A set of programs is described by which a consistent morphometry of bone sections can be performed. The programs are written in FORTRAN IV in a PDP 11/10 minicomputer, under the RT-11 operating system. The bone sections are run through the field of vision of the microscope using a scanning stage in one of several preselected courses at a magnification suitable for the parameters that are being put in. When input is completed, a print-out is produced with measured and derived parameters, personal data (protected by a code) and a comment file, all of which are stored for later retrieval and statistical evaluation. The programs were developed to be used in a system in which the bone sections are projected (by a projection microscope) onto a digitizer connected to the computer.

Follow-up of long-term treatment of predialysis renal bone disease with 1-alpha-hydroxy-derivatives of vitamin D.

Patients with predialysis chronic renal failure and bone disease were treated with 1 alpha-hydroxy-derivatives of vitamin D. The observation period consisted of 22 patient years. All patients showed histological improvement of renal osteodystrophy after the initial 6 months of treatment. Bone resorption indices improved most strongly. There was also a considerable decrease of non-mineralized osteoid and no change in the total trabecular bone volume. Hypercalcemia occurred 24 times in 9 patients. Although in general the 1,25-(OH)2-vitamin D serum levels were increased at the time of hypercalcemia, as compared to the preceding non-hypercalcemic period, no elevation above the normal range occurred. In cases of hypercalcemia two different groups of patients, with suppressed and non-suppressed levels of iPTH respectively, could be distinguished. In both groups different significant correlations between serum 1,25-(OH)2-vitamin D and serum calcium levels were found. If serum iPTH was not suppressed hypercalcemia was more severe. It is concluded that the occurrence and severity of hypercalcemia in patients with chronic renal failure during treatment with 1 alpha-hydroxy-derivatives of vitamin D is related to the presence of parathyroid hormone.