Anionic Effects on the Size and Shape of Apatite Crystals Grown from Physiological Solutions

Comparatively little is known of the role tissue fluid electrolytes have in establishing the size and shape of apatite crystals deposited in skeletal tissues. In vitro accretion experiments using synthetic apatite seed crystals comparable in size to bone apatite were performed to assess the extent to which these crystalline features may be affected by direct electrolyte/mineral interactions. A constant composition method was used to maintain the accretion reactions under physiological-like solution conditions (1.33 mmol/liter Ca²⁺, 1.0 mmol/liter total inorganic phosphate, (0 or 26) mmol/liter carbonate, 270 mmol/kg osmolality, pH 7.4, 37°C). When the mass of the new accretions equaled the initial seed mass, the solids were harvested and the net change in crystal size resulting from the new accretions was assessed by X-ray diffraction line-broadening analysis. All the electrolytes examined in this study inhibited the accretion rate. The order of effectiveness was phosvitin > polyaspartate ≈ polyglutamate > 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) > bovine serum albumin (BSA) > citrate. Citrate and BSA also reduced the mean crystal size of the harvested solids compared with those harvested in the absence of added electrolyte, a finding that suggests that citrate and BSA suppressed growth of the seed crystals in favor of the proliferation of new smaller crystals. In contrast, a net increase in size following accretion compared with controls suggests that the other more strongly inhibiting electrolytes stimulated growth of the primary seed crystals and/or of the secondary crystals. These size changes, however, were anisotropic, with the anions effecting primarily increases in crystal width/thickness rather than in length. The effects were also more pronounced in the presence of carbonate. Our findings suggest that the strength of the interaction with the crystal surface may be relatively more important than molecular size or conformational complexity in establishing the effect that electrolytes have on apatite growth and proliferation. The results also suggest that adsorbed electrolytes may be a significant factor in controlling the size of apatite crystals in skeletal tissues by inducing proliferation of new crystals and/or affecting crystal shape by selectively modifying growth of the lateral dimensions. Received: 28 June 1999 / Accepted: 3 January 2000     

The Effect of Fluoride on the Size and Morphology of Apatite Crystals Grown from Physiologic Solutions

In adult human bone, fluoride uptake is accompanied by an increase in apatite crystal size. This increase, however, is not isotropic but is restricted primarily to growth in width and/or thickness, with no measurable change in length. In the present study, seeded growth experiments were conducted in vitro to determine whether this anisotropic effect is physicochemical in origin, i.e., a direct result of F^- selectively enhancing lateral crystal growth, or is an indirect consequence of F^--induced alterations in cellular function and matrix development. The growth reactions were maintained at 37°C under physiologic-like solution conditions (1.33 mmol/liter Ca²⁺, 1.0 mmol/liter total phosphate, 0 or 26 mmol/liter carbonate, 270 mmol/kg osmolality, pH 7.4) using constant-composition methods. When new accretions accumulated to three times the initial seed mass, the solids were collected and net crystal growth was assessed by X-ray diffraction line broadening analysis. The X-ray results revealed that the carbonate constituent in our physiologic-like solutions promoted the proliferation of new crystals at the expense of further growth of the seed apatite. Solution F^- concentrations of ∼2 μmol/liter partially offset the repressive effect that carbonate had on primary crystal growth. Moreover, F^- stimulated seed crystal growth in the same anisotropic manner as had been observed for adult human bone apatite, a finding that suggests that the latter growth in vivo was the consequence, in part, of direct F^--mineral interactions. Received: 14 May 1997 / Accepted: 23 January 1998     

Stable Transfection of Nonosteogenic Cell Lines with Tissue Nonspecific Alkaline Phosphatase Enhances Mineral Deposition Both in the Presence and Absence of β-Glycerophosphate: Possible Role for Alkaline Phosphatase in Pathological Mineralization

It is documented that alkaline phosphatase (AP) plays an important role in bone mineralization. Considering that TN-AP is expressed in periodontal ligament fibroblasts, renal epithelial cells, and vascular endothelial cells, and that TN-AP is both a calcium-/phosphate-binding protein and a phosphohydrolytic enzyme, we hypothesize that membrane-bound AP also plays an important role in the initiation of physiological and pathological mineralizations in tissues other than bone and cartilage. To test this hypothesis, nonosteoblast cell lines, including a fibroblast line, a renal epithelial line, and a capillary endothelial line, were stably transfected to express high levels of rat bone AP on their cell surfaces. These rat bone AP-expressing cells were then cultured on filter membranes in the presence or absence of β-glycerol phosphate. von Kossa staining for calcium phosphate and transmission electron microscopy with electron diffraction analysis for minerals were employed to investigate the effect of membrane AP on extracellular calcium phosphate mineralization. Our results indicated that AP expression on these nonosteoblast-like cell surfaces have induced extracellular hydroxyapatite (HAP) mineralization. Our findings support the concept that membrane-bound AP contributes to extracellular apatitic mineralization by mechanisms that do not necessarily involve its hydrolase activity. They also suggest that AP might be important for the initiation of pathological mineralization in nonosteogenic tissues. Received: 11 January 1996 / Accepted: 31 October 1996     

Gene and Protein Expression During Differentiation and Matrix Mineralization in a Chondrocyte Cell Culture System

Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. Nasal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10⁵ cell/cm² and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and β-glycerophosphate. First, cells lost their phenotype but in this condition they rapidly reexpressed the chondrocyte phenotype and were able to form calcified cartilaginous nodules with the morphological appearance of cartilage mineralization that occurs in vivo during endochondral ossification. In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, proteoglycan core protein, characteristic markers of chondrocyte differentiation, as well as alkaline phosphatase and osteocalcin associated with the mineralization process. Analysis of labeled collagen and immunoblotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture. However, our culture conditions promoted extracellular matrix mineralization and cell differentiation towards the hypertrophic phenotype. This differentiation process was characterized by the induction of type X collagen mRNA, alkaline phosphatase, and diminished expression of type II collagen and core protein of large proteoglycan after an increase in their mRNA levels before the mineralizing process. These results revealed distinct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stages of the differentiation program in vitro. Received: 12 December 1996 / Accepted: 26 June 1997     

Biochemical Markers as Predictors of Bone Mineral Density Changes After GnRH Agonist Treatment

To evaluate bone biochemical markers as predictors of the efficacy of a hormone replacement therapy (HRT), we studied the bone changes induced by the cessation and return of ovarian function in 28 patients treated for 6 months with a GnRH agonist. This model reproduced the effects observed in postmenopausal women with high bone turnover treated with HRT. At the end of the treatment, Z scores were 1.8 ± 0.3 for Crosslaps (CTx) and deoxypyridinoline (D-Pyr), and 1.1 ± 0.2 for bone alkaline phosphatase (B-ALP) and osteocalcin (OC). This indicated an imbalance in bone remodeling with a high bone resorption. Bone mineral density (BMD) fell by 4.2 ± 2.5%. The changes in BMD between the 6th and 12th months were 0.34 ± 2.24 and −1.73 ± 3.25% at the lumbar spine and the femoral neck, respectively. Biochemical markers except urinary calcium and hydroxyproline measured at 6 months were positively correlated with the BMD changes at the lumbar spine. After the resumption of menstruation, 13 of 28 women displayed positive spine BMD changes between the 6th and 12th months; in this group, bone biochemical markers measured at 6 months were significantly higher (P= 0.02). Stepwise regression analysis showed that the association of B-ALP and D-Pyr measured at 6 months explained 40% of BMD variance and the association of B-ALP, PTH, and estradiol 56%. We conclude that measuring individual biochemical bone markers can help to predict the bone effect of an increase in the circulating estradiol in women with ovarian deficiency. Received: 16 January 1997 / Accepted: 17 June 1997     

Expression of BMP-2 by Rat Bone Marrow Stromal Cells in Culture

To investigate the role of bone morphogenetic protein (BMP-2) in ossifying rat bone marrow stromal cell cultures, we determined the population of fibroblast-like stromal cells that expressed BMP-2 immunocytochemically (anti-rhBMP-2 monoclonal antibody), and compared that to alkaline phosphatase (AP) and collagen synthesis formed in culture over a 4-week period in control and dexamethasone-supplemented mineralizing media. In control media, the percentage of BMP-2-positive stromal cells (BMP-2⁺) increased from 12 to 25% within the first 4 days of culture. In mineralizing media, the level of BMP-2⁺ cells was significantly increased (43–44%). The intensity of immunostaining gradually increased with time. The levels of AP were undetectable at 1 week in both control and mineralizing media, but increased gradually over the next 2 weeks and peaked at 3 weeks. ALP levels were significantly greater in cultures grown in mineralizing medium (P < 0.05 at 3 weeks, P < 0.01 at 4 weeks). Collagen synthesis peaked and was significantly greater at 3 weeks (P < 0.05) in cultures grown in mineralizing medium. The levels of AP and collagen synthesis most closely reflected the changes in the percentage of BMP-2⁺ cells from 7 to 28 days. Though these changes may reflect a primary action of BMP-2 on marrow osteoprogenitor-like stromal cells, they do not exclude a mechanism that involves the induction of other members of the BMP family known to stimulate AP and collagen synthesis. We conclude that BMP-2 expression in cultures of fibroblast-like marrow stromal cells is enhanced when those cells are induced to become osteoblasts by exposure to dexamethasone. Received: 30 October 1997 / Accepted: 24 June 1998     

Resistive Training Maintains Bone Mineral Density in Postmenopausal Women

We examined the effects of a total body resistive training program (RT) on total and regional bone mineral density (BMD) in older women. Twenty-seven healthy postmenopausal women (mean age 62 ± 1 years) participated in a strength training program three times/week for 16 weeks. Strength was assessed before and after training by either one or three repetition maximum (1RM and 3RM) tests. Both upper and lower body strength significantly increased by 36–65% and 32–98%, respectively, after training. There was a small but significant decrease in body weight and body mass index after training (P < 0.05), with no change in the waist-to-hip ratio. BMD, assessed by dual-energy X-ray absorptiometry, did not change over the duration of the training period in the anterioposterior spine (L₂–L₄), femoral neck, Ward's triangle, and greater trochanter. BMD of the total body, lateral spine (B₂–B₄), and the regions of the radius (1/3 radius and ultradistal radius) also did not fall in subsets of these women. Muscular strength of both the leg and chest press were significantly associated with L₂–L₄, femoral neck, Ward's triangle, and greater trochanter BMD (range r = 0.57–0.84, all P < 0.005). Markers of bone turnover, namely, bone-specific alkaline phosphatase, osteocalcin, and urinary aminoterminal cross-linked telopeptide of type I collagen did not change significantly. In conclusion, a resistive training program maintains BMD and improves muscular strength in healthy, older women. This may be important in preventing the negative health outcomes associated with the age-related loss of bone density. Received 5 June 1996 / Accepted: 26 June 1997     

The Effect of Fluoride Treatment on Bone Mineral in Rabbits

Fluoride therapy has been used clinically for many years, but its use remains controversial and many basic questions remain unanswered. Accordingly, this study returns to an animal model to study the effects of high doses of fluoride on bone mineral in rabbits. Twelve rabbits, aged 3(1/2) months at the start of the study, received drinking water fluoridated at 100 ppm while their 12 control counterparts drank distilled water. All rabbits were sacrificed after 6 months. Fluoride was readily incorporated into femoral cortical bone (7473 +/- 966 ppm F versus 1228 +/- 57 ppm in controls; P < 0.00005). Fluoride therapy led to increased mineralization, as measured by density fractionation (P < 0.0005 for the distributions). The bone mineral itself was altered, with a significant increase in the width of crystals (66.2 +/- 2.0 A versus 61.2 +/- 0.9 A; P < 0.01). The microhardness of both cortical and cancellous bone in the femoral head of fluoride-treated rabbits was greater than that in the controls (P < 0.05). The phosphate, calcium, and carbonate contents in the bone was the same in both groups. Finally, fluoride administration did not affect the architecture or connectivity of cancellous bone in the femoral head. Previously published data [1] indicated that the mechanical properties of bone were adversely affected; this suggests that the effect of high doses of fluoride on the strength and stiffness of bone may be mediated by its effect on bone mineral.     

Bone Mineral Content and Density in Professional Tennis Players

Total and regional bone mineral content (BMC) as well as lean and fat mass were measured in nine male professional tennis players (TPs) and 17 nonactive subjects; dual-energy X-ray absorptiometry (DXA) was used for measuring. The mean (±SD) age, body mass, and height were 26 ± 6 and 24 ± 3 years, 77 ± 10 and 74 ± 9 kg, and 180 ± 6 and 178 ± 6 cm for the TP and the control group (CG), respectively. The whole body composition for BMC, lean mass, and fat of the TP was similar to that observed in the CG. The tissue composition of the arms and legs was determined from the regional analysis of the whole-body DXA scan. The arm region included the hand, forearm, and arm, and was separated from the trunk by an inclined line crossing the scapulo-humeral joint. In the TP, the arm tissue mass (BMC + fat + lean mass) was about 20% greater in the dominant compared with the contralateral arm because of a greater lean (3772 ± 500 versus 3148 ± 380 g, P < 0.001) and BMC (229.0 ± 43.5 versus 188.2 ± 31.9 g, P < 0.001). In contrast, no significant differences were observed either in BMC or BMD between arms in the CG. Total mass, lean mass, and BMC were greater in the dominant arm of the TP than in the CG (all P < 0.05). In the TP, BMD was similar in both legs whereas in the CG, BMD was greater in the right leg. Lumbar spine (L2–L4) BMD, adjusted for body mass and height, was 15% greater in the TP than in the CG (P < 0.05). Femoral neck BMDs (femoral neck, Ward's triangle, greater trochanter, and intertrochanteric regions) adjusted for body mass and height were 10–15% greater in the TP (all P < 0.05). Ward's triangle BMD was correlated with the maximal leg extension isometric strength (r = 0.77, P < 0.05) even when adjusted for body mass (r = 0.76, P < 0.05) and height (r = 0.77, P < 0.05). In summary, the participation in tennis is associated with increased BMD in the lumbar spine and femoral neck. These results may have implications for devising exercise strategies in young and middle-aged persons to prevent involutional osteoporosis later in life. Received: 29 April 1997 / Accepted: 14 November 1997     

X-Ray Diffraction on Cyclically Loaded Osteons

The results of a study on the fine structural distortion due to the two previously observed types of degradation in cyclically loaded single osteons (i.e., stiffness degradation and pinching effect) are presented. Fully calcified longitudinal and alternate osteons were isolated from 350-μm-thick longitudinal sections of human femoral cortical bone. The samples were prepared from 500-μm-long central cylindrical portions of an osteon, whose two ends were penetrating into rectangular lugs for fixation to an electromechanical device that cyclically loaded the samples. This device was connected to a microwave micrometer and a recorder. The structural distortions induced by cyclic loading were investigated by high- and low-angle X-ray diffraction on conventional and synchrotron radiation sources. Cyclic loading results in a reduction in the degree of orientation of apatite crystallites, especially in longitudinal osteons, in which the most abundant longitudinal lamellae are not protected against buckling by transverse lamellae as they are in alternate osteons. In contrast, the degree of orientation of collagen fibrils does not seem to be affected by cycling loading in the two osteon types, possibly because the disorientation of collagen fibrils is, within limits, a reversible process. Finally, the contrast between the disorientation of inorganic crystallites and the apparently unaltered distribution of collagen fibrils suggests that the degradation of cyclically loaded osteons may be due to a separation of the crystallites from the fibrils. Received: 6 June 1996 / Accepted: 7 August 1997     

Bioglass ®45S5 Stimulates Osteoblast Turnover and Enhances Bone Formation In Vitro: Implications and Applications for Bone Tissue Engineering

We investigated the concept of using bioactive substrates as templates for in vitro synthesis of bone tissue for transplantation by assessing the osteogenic potential of a melt-derived bioactive glass ceramic (Bioglass 45S5) in vitro. Bioactive glass ceramic and bioinert (plastic) substrates were seeded with human primary osteoblasts and evaluated after 2, 6, and 12 days. Flow cytometric analysis of the cell cycle suggested that the bioactive glass-ceramic substrate induced osteoblast proliferation, as indicated by increased cell populations in both S (DNA synthesis) and G2/M (mitosis) phases of the cell cycle. Biochemical analysis of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin indicated that the bioactive glass-ceramic substrate augmented osteoblast commitment and selection of a mature osteoblastic phenotype. Scanning electron microscopic observations of discrete bone nodules over the surface of the bioactive material, from day 6 onward, further supported this notion. A combination of fluorescence, confocal, transmission electron microscopy, and X-ray microprobe (SEM-EDAX) examinations revealed that the nodules were made of cell aggregates which produced mineralized collagenous matrix. Control substrates did not exhibit mineralized nodule formation at any point studied up to 12 days. In conclusion, this study shows that Bioglass 45S5 has the ability to stimulate the growth and osteogenic differentiation of human primary osteoblasts. These findings have potential applications for tissue engineering where this bioactive glass substrate could be used as a template for the formation of bioengineered bone tissue.     

Diurnal Variation of Bone Mineral Turnover in Elderly Men and Women

The diurnal variation of markers of bone mineral metabolism have been documented in pre- and early postmenopausal women. Such rhythms have clinical implications for timing of sample collection and assessment of therapeutic intervention. To examine the diurnal variation of bone turnover in the elderly, we examined markers of bone formation [serum osteocalcin (OC) and bone-specific alkaline phosphatase (B-ALP)]; a marker of bone resorption (urinary N-telopeptide cross-linked collagen type 1 [NTX]); and serum calcium and parathyroid hormone (PTH) over 24 hours. Subjects were healthy community-dwelling elderly who were on no medications known to significantly alter bone mineral metabolism. Subjects included 14 women [74 ± 6 years (mean ± SD)] and 14 men (80 ± 5 years). Over the 24-hour sampling period, mean serum OC, B-ALP, and calcium values were similar in elderly men and women. However, mean serum PTH was significantly higher in elderly men compared with women (P < 0.05). The magnitude of the diurnal variation of urinary NTX was significantly higher in women compared with men (P < 0.05). There was a significant diurnal variation for serum OC, B-ALP, calcium, PTH, and urinary NTX in both elderly men and women. The magnitude of the diurnal variation was approximately 10–20% of mean value for OC and B-ALP, 30% for PTH, and up to 40% for urinary NTX. We conclude that there is significant diurnal variation in the markers of bone mineral metabolism for elderly men and women. The peak value, which on average would be 20% higher than the mean value for urinary NTX, highlights the importance of the timing of sample collection for appropriate interpretation of therapeutic response. In addition, gender-related differences, including relatively higher levels of serum PTH and lower levels of urinary NTX in elderly men, may help explain differences in rates of bone loss in this age group. Received: 21 June 1996 / Accepted: 18 October 1996     

Bone Mineral Density and Androgen Levels in Elderly Males

To clarify the relationship of sex male hormones and bone in men, we studied in 140 healthy elderly men (aged 55–90 years) the relation between serum levels of androgens and related sex hormones, bone mineral density (BMD) at different sites, and other parameters related to bone metabolism. Our results show a slight decrease of serum-free testosterone with age, with an increase of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in a third of the elderly subjects studied. BMD decreased significantly with age in all regions studied, except in the lumbar spine. We found a positive correlation between body mass index (BMI) and BMD at the lumbar spine and femoral neck (P < 0.001). No relationship was found (uni- and multivariate regression analysis) between serum androgens or sex hormone-binding globulin (SHBG) and BMD. We found a positive correlation of vitamin D binding protein (DBP) and osteocalcin with lumbar spine BMD and with BMI, DBP, IGF-1, and PTH with femoral neck BMD. In conclusion, there is a slight decline in free testosterone and BMD in the healthy elderly males. However, sex male hormones are not correlated to the decrease in hip BMD. Other age-related factors must be associated with bone loss in elderly males. Received: 29 April 1997 / Accepted: 9 November 1997     

Osteoporosis and Bone Mineral Metabolism Disorders in Cirrhotic Patients Referred for Orthotopic Liver Transplantation

The purpose of this study was to determine the prevalence of osteoporosis, to estimate the bone turnover and hormonal status, and to identify the factors associated with bone disease in patients with end-stage liver disease who were referred for orthotopic liver transplantation. A prospective study was performed on 58 cirrhotic patients (6 with primary biliary cirrhosis, 14 with alcoholic cirrhosis, and 38 with posthepatitic cirrhosis), who were referred for orthotopic liver transplantation. Patients, excluding those with primary biliary cirrhosis, were classified in Child-Pugh groups according to the severity of liver disease (class B [28 patients], class C [24 patients]). Biochemical parameters of bone mineral metabolism and standard liver function tests were measured in all patients. Additionally, serum osteocalcin, urinary hydroxyproline/creatinine ratio, serum intact parathyroid hormone, serum 25-hydroxyvitamin D, serum 1,25-dihydroxyvitamin D, folliclestimulating hormone, and luteinizing hormone levels were determined in patients and controls within the same age range. Plasma testosterone, sex hormone-binding globulin levels, and free testosterone index were obtained for all men included in the study. Bone mass of the lumbar spine and femur were measured by dual X-ray absorptiometry (DPX-L), and were expressed as a standard deviation of mean values (Z-score) from a sex and age-matched control group. Spinal X-rays were obtained to assess vertebral fractures. Osteoporosis was considered as a factor in spinal bone mineral density with a Z-score below 2 or at least one vertebral fracture. Twenty-five patients (43%) had osteoporosis, with lower bone mass measurements in the lumbar spine than in the femoral neck (P < 0.005). Alcoholic and Child-Pugh C patients showed the lowest femoral bone mineral density values. Cirrhotic patients showed lower osteocalcin levels than controls (14.3 ± 5.9 vs. 18.2 ± 8.1 ng/ml; P < 0.05) and showed increased urinary hydroxyproline (125.1 ± 51.5 vs. 107.9 ± 26.6 nM/mg creatinine; P < 0.05). Serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone levels were significantly lower in cirrhotic patients than in controls (10.3 ± 9.1 vs. 23.1 ± 26.6 ng/ml; P= 0.000), (12.9 ± 9.1 vs. 48.3 ± 11.5 pg/ml; P= 0.000), (16.6 ± 9.2 vs. 27.9 ± 8.2 pg/ml; P= 0.000), with no differences between Child-Pugh groups. Alcoholic Child-Pugh C patients showed the lowest 25-hydroxyvitamin D serum values (4.5 ± 2.2 ng/ml; P < 0.05). Male patients had lower testosterone levels than controls (302.5 ± 229.4 vs. 556.7 ± 146.5 ng/dl; P= 0.000), with increased sex hormone-binding globulin values. Levels of testosterone and gonadotropin were related to Child-Pugh classification. No correlation was found between bone mass and hormonal values. A significant decrease in bone mass, particularly in the lumbar spine, is seen in end-stage cirrhotic patients. Reduced bone formation and significant disorders of bone mineral metabolism, such as vitamin D deficiency, reduced parathyroid hormone levels, and hypogonadism are involved. Moreover, severity and etiology of the liver disease are the main risk factors for developing bone loss and mineral metabolism disorders in patients referred for orthotopic liver transplantation. Received: 7 March 1996 / Accepted: 24 June 1996     

Loss of Bone Mineral Density in Women Athletes During Aging

Total and regional bone mineral density (BMD) by dual-energy-X-ray absorptiometry (DXA) and bone turnover were tested in 50 highly trained women athletes and 21 sedentary control women (18–69 years; BMI < 25 kg/m²). VO_2max (ml · kg⁻¹· min⁻¹) and lean tissue mass (DXA) were significantly higher in the athletes versus controls (both P < 0.0001). Total body BMD did not decline significantly with age in the athletes whereas lumbar spine (L₂–L₄) BMD approached statistical significance (r =−0.26; P= 0.07). Significant losses of the femoral neck (r =− 0.42), Ward's triangle (r =−0.53), and greater trochanter BMD (r =−0.33; all P < 0.05) occurred with age in the athletes. In the athletes, total body BMD, L₂–L₄ BMD, and BMD of all sites of the femur were associated with lean tissue mass (r = 0.32 to r = 0.57, all P < 0.05) and VO_2max (r = 0.29 to r = 0.48, all P < 0.05). Femoral neck and greater trochanter BMD were higher in the athletes than in controls (both P < 0.05) and lumbar spine and Ward's triangle BMD approached statistical significance (both P= 0.07). Bone turnover was assessed by serum bone-specific alkaline phosphatase (B-ALP), urinary deoxypyridinoline cross-links (Dpd), and urinary aminoterminal cross-linked telopeptides (NTX). There were no relationships between B-ALP or Dpd with age whereas NTX increased with age (r = 0.46, P < 0.05) in the entire group. Levels of B-ALP and NTX were negatively associated with total body, L₂–L₄, femoral neck, Ward's triangle, and greater trochanter BMD (P < 0.05). B-ALP and Dpd were not significantly different between athletes and controls whereas NTX was lower in the athletes than in controls (P < 0.001). The high levels of physical activity observed in women athletes increase aerobic capacity and improve muscle mass but are not sufficient to prevent the loss of bone with aging. Received: 28 November 1997 / Accepted: 8 April 1998     

Bone Mineral Density Is a Predictor of Survival

The purpose of this study was to examine the relationship between bone mineral density (BMD) and survival in both sexes and to compare BMD with other established risk factors such as blood pressure and cholesterol. A population-based prospective study of 1924 individuals (850 men, 1074 women) was performed in G?teborg from 1980 to 1983. Measurements of BMD were obtained in 1468 (76%) of the participants (653 men, 815 women). This selection of individuals generated 10,965 person years, and death was registered for 289 men and 197 women in the 7-year period (2661 days) after bone mineral measurement. Later information on date of death was obtained from the official population register. This information covers 7 years from the time of survey of the last examined participant (in Dec. 1983). At the beginning of the study, BMD was measured in the calcaneus by dual photon absorptiometry (DPA), and blood pressure, serum cholesterol, serum triglycerides, and body mass index (BMI) were also recorded. The study was coordinated with the National Register of Causes of Death and the National Cancer Register. A modified version of the Cox proportional hazards model was used to calculate and determine the age-adjusted relations between nontrauma mortality and BMD. When the various quartiles of BMD were compared prospectively from 70, 75, and 79 years of age with survival figures during the 2661-day follow-up period, the first and the second quartiles with the lowest BMD at entry showed the lowest survival rate in both men (P= 0.01) and women (P= 0.01). A decrease of 1 SD of BMD in a univariate analysis was associated with a 1.39-fold increase in mortality in both men (95% confidence interval 1.25–1.56, P < 0.001) and women (95% confidence interval 1.22–1.58, P < 0.001), and a multivariate analysis demonstrated a relative risk of 1.23 (95% confidence interval 1.10–1.41, P < 0.001) in men and 1.19 (95% confidence interval 1.02 to 1.39, P= 0.019) in women. All relations were adjusted for sex, age, and follow-up. This study indicates that BMD is a predictor of survival, especially for subjects over 70. Bone mineral density was found to be a better predictor of death than blood pressure and cholesterol. This study indicates that, after adjustments have been made for diseases, low bone mass is an independent predictor of mortality and might be a marker of general health or functional aging. Its measurement might therefore be a valuable tool in general health investigations. Received: 26 December 1996 / Accepted: 27 January 1998     

Metastable Equilibrium Solubility Behavior of Bone Mineral

Previous studies have shown that carbonated apatites with a range of carbonate contents and crystallinities exhibit the phenomenon of metastable equilibrium solubility (MES) distributions. The purpose of the present study was to investigate the solubility behavior of bone mineral using the concepts of MES and MES distributions and, together with crystallinity and chemical composition data, examine the similarity of bone mineral to carbonated apatite (CAP). Bone samples were harvested from 1-, 5-, and 8-month-old rats. The organic components of the bone samples were removed by hydrazine deproteination. Carbonated apatite was synthesized by the hydrolysis of dicalcium phosphate dihydrate (DCPD) in a NaHCO₃-containing media at 50°C. The MES distributions of bone mineral and CAP were determined by equilibrating predetermined amounts of CAP or bone mineral in a series of 0.1 M acetate buffers containing calculated levels of calcium and phosphate and maintained at essentially constant pHs of 5.0, 5.3, 5.7, and 6.5. From the compositions of the equilibrating buffer solutions, ion activity products based upon the stoichiometries of octacalcium phosphate, hydroxyapatite, and carbonated apatite were calculated in an attempt to determine the function governing the dissolution of CAP and bone mineral. The results of this study demonstrated that the MES distribution phenomenon appeared to hold for bone mineral and that the changes in crystallinity of bone mineral with age correlated well with changes in the MES values. A CAP sample was prepared that was found to be an excellent synthetic prototype closely mimicking the physicochemical behavior of bone mineral from an 8-month-old rat. Another finding of this study was that the ion activity product function based upon the hydroxyapatite stoichiometry well described the MES results obtained with both CAP and bone mineral. The interpretation that a surface complex with hydroxyapatite stoichiometry governs the solubility behavior of bone mineral is, therefore, consistent with the experimental data. Other calcium phosphate stoichiometries for the surface complex showed systematic variations in the MES profiles when the pH of the equilibrating solution was varied. Received: 30 October 1997 / Accepted: 1 October 1998     

Bone Mineral Density in Flatwater Sprint Kayakers

To elucidate the possible skeletal benefits of the muscular contractions and the nonweight-bearing loading pattern associated with kayaking, we investigated the bone mineral density (BMD, g/cm²) of 10 elite kayakers, six males and four females, with a median age of 19 years. Each subject was compared with the mean value of two matched controls. BMD of the total body, head, ribs, humerus, legs, proximal femur (neck, wards, trochanter), spine, lumbar spine, and bone mineral content (BMC, g), of the arms was obtained using a dual energy X-ray absorptiometer (DXA). Body composition was also assessed. The kayakers had a significantly (P < 0.05–0.01) greater BMD in most upper body sites: left and right humerus (10.4% and 11.7%), respectively, ribs (6.4%), spine (10.9%), and a greater BMC of the left and right arm (15.7% and 10.6%, respectively). No significant differences in the BMD of the total body, head, or any of the lower body sites were found, except for the pelvis, which was significantly greater in kayakers (5.1%). The controls had a significantly lesser lean body mass (10.4%) and greater percentage of body fat (19.5%) than the kayakers. Bivariate correlation analysis in the controls demonstrated significant and strong relationships between BMD in upper body sites and lean body mass, weight, and fat; the effects of training seem to outweigh most such relationships in kayakers. In conclusion, it seems that the loading pattern and muscular contractions associated with kayaking may result in site-specific adaptations of the skeleton. Received: 21 April 1998 / Accepted: 1 October 1998     

Bone Mineral Density in the Chronic Patellofemoral Pain Syndrome

Bone mineral density (BMD) and clinical status of 40 patients with a chronic, unilateral patellofemoral pain syndrome (PFPS) were determinated. The mean duration of the disease at the time of the follow-up was 7.6 ± 1.8 (SD) years. The BMD was measured at the spine (L2–L4), and the femoral neck, trochanter area of the femur, distal femur, patella, proximal tibia, and calcaneus of both lower extremities using a dual-energy X-ray absorptiometric (DXA) scanner. The mean BMD of the affected limb (compared with the unaffected side) was significantly lower in the distal femur (−3.3%; P= 0.002), patella (−2.5%; P= 0.016), and proximal tibia (−1.9%; P= 0.008). The femoral neck, trochanter area of the femur, and calcaneus showed no significant side-to-side differences, and the spinal BMDs of men and women with the PFPS were comparable with the manufacturer's age-adjusted reference values for Western European men and women. The relative BMDs of the affected knee showed strongest correlation with the muscle strength of the same knee: the better the muscle strength compared with the healthy knee, the higher the relative BMD (r = 0.56–0.58 with P < 0.001 in each anatomic site of the knee). In the stepwise regression analysis, low body weight or low body mass index, high level of physical activity, the patient's good subjective overall assessment of his/her affected knee, and short duration of the symptoms were also independent predictors of the high relative BMD in the affected knee so that along with the muscle strength these variables could account for 51% of the variation seen in the relative BMD of the femur, 61% in the patella, and 54% in the proximal tibia. In conclusion, chronic patellofemoral pain syndrome results in a significantly decreased BMD in the knee region of the affected limb. The spine, proximal femur, and calcaneus are not affected. Recovery of normal muscle strength and knee function seems to be of great importance for good BMD. Received: 30 May 1997 / Accepted: 8 January 1998