The effects of visceral obesity and androgens on bone: trenbolone protects against loss of femoral bone mineral density and structural strength in viscerally obese and testosterone-deficient male rats.

SUMMARY: In males, visceral obesity and androgen deficiency often present together and result in harmful effects on bone. Our findings show that both factors are independently associated with adverse effects on femoral bone structure and strength, and trenbolone protects rats from diet-induced visceral obesity and consequently normalises femoral bone structural strength. INTRODUCTION: In light of the rapidly increasing incidence of obesity and osteoporosis globally, and recent conjecture regarding the effects of visceral adiposity and testosterone deficiency on bone health, we investigated the effects of increased visceral adipose tissue (VAT) mass on femoral bone mineral density (BMD), structure and strength in normal weight rats with testosterone deficiency. METHODS: Male Wistar rats (n = 50) were fed either standard rat chow (CTRL, n = 10) or a high-fat/high-sugar diet (HF/HS, n = 40). Following 8 weeks of feeding, rats underwent sham surgery (CTRL, n = 10; HF/HS, n = 10) or orchiectomy (HF/HS + ORX, n = 30). Following a 4-week recovery period, mini-osmotic pumps containing either vehicle (CTRL, n = 10; HF/HS, n = 10; HF/HS + ORX, n = 10), 2.0 mg kg day(-1), testosterone (HF/HS + ORX + TEST, n = 10) or 2.0 mg kg day(-1) trenbolone (HF/HS + ORX + TREN, n = 10) were implanted for 8 weeks of treatment. Dual-energy X-ray absorptiometry and three-point bending tests were used to assess bone mass, structure and strength of femora. RESULTS: Diet-induced visceral obesity resulted in decreased bone mineral area (BMA) and content (BMC) and impaired femoral stiffness and strength. Orchiectomy further impaired BMA, BMC and BMD and reduced energy to failure in viscerally obese animals. Both TEST and TREN treatment restored BMA, BMC, BMD and energy to failure. Only TREN reduced visceral adiposity and improved femoral stiffness and strength. CONCLUSIONS: Findings support a role for both visceral adiposity and testosterone deficiency as independent risk factors for femoral osteoporosis, adverse bone geometry and impaired bone strength in male rats. Trenbolone may be a more effective candidate for androgen replacement therapy than testosterone in viscerally obese testosterone-deficient males.

MCP-1 expression is specifically regulated during activation of skeletal repair and remodeling.

Monocyte chemotactic protein-1 (MCP-1) belongs to the CC chemokine superfamily and plays a critical role in the recruitment and activation of leukocytes during acute inflammation. We hypothesize that MCP-1 is also an important chemokine that regulates the recruitment and activation of bone cells required for skeletal repair and remodeling. We used the ulnar stress fracture (SFx) model, which allows investigation of focal remodeling with a known time course and precise anatomical location. SFx were created in the right ulna of female Wistar rats using cyclic end loading. Unloaded animals were used as a control. Rats were killed 4 h and 1, 4, 7, and 14 days after loading (n = 10/group); RNA was extracted and converted to cDNA for quantitative PCR analysis using TaqMan gene expression assays. Four hours after loading, MCP-1 gene expression was increased ~30-fold (P < 0.001), remained elevated at 24 h (~12-fold, P < 0.001), then declined by day 14. Relative to the contralateral limb, expression of the receptors CCR1 and CCR2 increased over the 14 days, being significant by 4 days for CCR1 and 14 days for CCR2 (P < 0.05). Other inflammation-related chemokines (RANTES, MIP1a) were not increased at these early time points. Using in situ hybridization and immunohistochemistry in separate animal groups (n = 5/group, control, days 1, 4, 7), MCP-1 mRNA and protein were localized in periosteal osteoblasts associated with woven bone formation at the fracture exit point but not in osteocytes adjacent to the SFx. These data support an important role for MCP-1 in the early phase of SFx repair and activated remodeling.

Translational aspects of bone quality--vertebral fractures, cortical shell, microdamage and glycation: a tribute to Pierre D. Delmas.

Among vertebral deformities, the prevalence of wedge fractures is about twice that of endplate (biconcave) deformities, both of which are greater than that of crush deformities. The anterior cortex is, therefore, a site of interest for understanding mechanisms of vertebral fracture. Despite its importance to vertebral mechanics, there are limited data describing the role of cortical shell, microdamage, and bone matrix parameters in vertebral fragility. This review of literature emphasizes the translational aspects of bone quality and demonstrates that a greater understanding of bone fractures will be gained through bone quality parameters related to both cortical and cancellous compartments as well as from microdamage and bone matrix parameters. In the context of vertebral fractures, measures of cortical shell and bone matrix parameters related to the organic matrix (advanced glycation products and alpha/beta CTX ratio) are independent of BMD measurements and can therefore provide an additional estimate of fracture risk in older patients.

Inducible cyclo-oxygenase (COX-2) mediates the induction of bone formation by mechanical loading in vivo.

In vivo, indomethacin blockade of bone formation has been used to illustrate the role of prostaglandins. Indomethacin blocks the constitutive (COX-1) and inducible (COX-2) forms of cyclo-oxygenase, and is therefore nonspecific in its action. To test the hypothesis that COX-2 mediates the bone formation response to loading, rats were treated with vehicle, NS-398 (a specific COX-2 inhibitor) or indomethacin at 0.02, 0.2, or 2.0 mg/kg p.o. 3 h before loading the right tibia in four-point bending. Bending or sham loads of 65 N were applied for one bout of 300 cycles and bone formation assessed 5-8 days after loading. Mechanically induced bone formation at the endocortical surface was calculated by subtracting formation indices of the left leg (control) from those of the right (loaded), and woven bone surface and area were measured at the periosteal surface. Endocortical bone formation was significantly increased by bending but not sham loading (p < 0.05). The increase in the endocortical bone formation rate and mineralizing surface caused by bending was only partially inhibited by indomethacin, even at the highest dose, whereas NS-398 completely blocked bone formation at all doses (p < 0.05). The mineral apposition rate was depressed in a dose-response fashion by NS-398 (p < 0.05), but not by indomethacin. Woven bone formation at the periosteal surface was not prevented by treatment with indomethacin nor NS-398, suggesting that its formation is not dependent on prostaglandin production. These data suggest that induction of COX-2 is important for lamellar bone formation elicited by mechanical strain.

Mechanical loading thresholds for lamellar and woven bone formation.

Bone formation was measured in rat tibiae after 12 days of applied loading. Bending forces were applied using a four-point loading apparatus. Sham loads were applied at the same magnitudes as bending forces but the loading pads were arranged so that bending was minimized. Bending and sham loading were applied to the right tibiae of rats and the left tibiae served as contralateral controls. Loading was applied as a sine wave with a frequency of 2 Hz for 18 s (36 cycles) per day. The peak magnitude of applied load was 27, 33, 40, 52, and 64 N. Woven bone was observed on the periosteal surface in all animals subjected to loads of 40 N or greater. Periosteal woven bone formation occurred in both bending and sham loading groups. Woven bone formation on the periosteal surface was either absent or responded at a maximal rate if the stimulus threshold was surpassed. The amount of new woven bone and the woven bone-forming surface were independent of the magnitude of applied strain. Bone formation on the endocortical surface was exclusively lamellar. Lamellar bone formation was stimulated by applied bending of the tibia but not by sham loading. Bending strains above a loading threshold of 40 N or about 1050 mu strain increased both bone-forming surface and the mineral apposition rate and subsequently increased the bone formation rate as much as sixfold. No evidence of increased bone formation was seen for applied strains below 1050 mu strain. Examination of bulk stained sections from animals exposed to the highest applied loads showed no evidence of microcracks.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone is permissive for skeletal adaptation to mechanical loading.

The Lewis dwarf (DW) rat was used as a model to test the hypothesis that growth hormone (GH) is permissive for new bone formation induced by mechanical loading in vivo. Adult female Lewis DW rats aged 6.2 +/- 0.1 months (187 +/- 18 g) were allocated to four vehicle groups (DW), four GH treatment groups at 32.5 microg/100 g body mass (DWGH1), and four GH treatment groups at 65 microg/100 g (DWGH2). Saline vehicle or GH was injected intraperitoneally (ip) at 6:30 p.m. and 6:30 a.m. before mechanical loading of tibias at 7:30 a.m. A single period of 300 cycles of four-point bending was applied to right tibias at 2.0 Hz, and magnitudes of 24, 29, 38, or 48N were applied. Separate strain gauge analyses in 5 DW rats validated the selection of loading magnitudes. After loading, double-label histomorphometry was used to assess bone formation at the periosteal surface (Ps.S) and endocortical surface (Ec.S) of tibias. Comparing left (unloaded) tibias among groups, GH treatment had no effect on bone formation. Bone formation in tibias in DW rats was insensitive to mechanical loading. At the Ec.S, mechanically induced lamellar bone formation increased in the DWGH2 group loaded at 48N (p < 0.05), and no significant increases in bone formation were observed among other groups. The percentage of tibias expressing woven bone formation (Wo.B) at the Ps.S was significantly greater in the DWGH groups compared with controls (p < 0.05). We concluded that GH influences loading-related bone formation in a permissive manner and modulates the responsiveness of bone tissue to mechanical stimuli by changing thresholds for bone formation.

Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib.

It has been hypothesized that suppression of bone remodeling allows microdamage to accumulate, leading to increased bone fragility. This study evaluated the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties of cortical bone in the dog rib. Thirty-six female beagles, 1-2 years old, were divided into three groups. The control group (CNT) was treated daily for 12 months with saline vehicle. The remaining two groups were treated daily with risedronate (RIS) at a dose of 0.5 mg/kg per day or alendronate (ALN) at 1.0 mg/kg per day orally. After sacrifice, the right ninth rib was assigned to cortical histomorphometry or microdamage analysis. The left ninth rib was tested to failure in three-point bending. Total cross-sectional bone area was significantly increased in both RIS and ALN compared with CNT, whereas cortical area did not differ significantly among groups. One-year treatment with RIS or ALN significantly suppressed intracortical remodeling (RIS, 53%; ALN, 68%) without impairment of mineralization and significantly increased microdamage accumulation in both RIS (155%) and ALN (322%) compared with CNT. Although bone strength and stiffness were not significantly affected by the treatments, bone toughness declined significantly in ALN (20%). Regression analysis showed a significant nonlinear relationship between suppressed intracortical bone remodeling and microdamage accumulation as well as a significant linear relationship between microdamage accumulation and reduced toughness. This study showed that suppression of bone turnover by high doses of bisphosphonates is associated with microdamage accumulation and reduced some mechanical properties of bone.

The ratio of messenger RNA levels of receptor activator of nuclear factor kappaB ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis.

The determinants of cancellous bone turnover and trabecular structure are not understood in normal bone or skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor kappaB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Enhanced expression of osteocalcin mRNA in human osteoarthritic trabecular bone of the proximal femur is associated with decreased expression of interleukin-6 and interleukin-11 mRNA.

Few studies have investigated the factors or mechanisms that may lead to structural changes in OA bone. This study examines the in vivo expression of messenger RNA encoding the osteoclastogenic cytokines interleukin-6 (IL-6) and interleukin-11 (IL-11), together with the osteoblastic marker osteocalcin (OCN) and the calcitonin receptor (CTR), which in bone is exclusively expressed by osteoclasts. Total RNA was isolated from intertrochanteric trabecular bone from OA patients, and from controls taken at autopsy. The patterns of mRNA expression of IL-6, IL-11, OCN, and CTR were examined using reverse-transcription polymerase chain reaction (RT-PCR) by determining the relative ratios of the amplified products with respect to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both IL-6 and IL-11 mRNA were significantly less abundant in OA than in the control group. Expression of IL-11 mRNA decreased significantly with age for both groups. OCN mRNA expression was significantly more abundant in OA, and there was no significant difference for CTR mRNA between the two groups. For both OCN and CTR in OA, expression increased significantly with increasing age. These differences in expression between the OA and control groups are consistent with an hypothesis that biochemical and genetic factors in bone can contribute or perhaps underlie the degenerative joint changes seen in OA.

Transgenic mice overexpressing tartrate-resistant acid phosphatase exhibit an increased rate of bone turnover.

Tartrate-resistant acid phosphatase (TRAP) is a secreted product of osteoclasts and a lysosomal hydrolase of some tissue macrophages. To determine whether TRAP expression is rate-limiting in bone resorption, we overexpressed TRAP in transgenic mice by introducing additional copies of the TRAP gene that contained the SV40 enhancer. In multiple independent mouse lines, the transgene gave a copy number-dependent increase in TRAP mRNA levels and TRAP activity in osteoclasts, macrophages, serum, and other sites of normal low-level expression (notably, liver parenchymal cells, kidney mesangial cells, and pancreatic secretory acinar cells). Transgenic mice had decreased trabecular bone consistent with mild osteoporosis. Measurements of the bone formation rate suggest that the animals compensate for the increased resorption by increasing bone synthesis, which partly ameliorates the phenotype. These mice provide evidence that inclusion of an irrelevant enhancer does not necessarily override a tissue-specific promoter.

Bone microdamage and skeletal fragility in osteoporotic and stress fractures.

The accumulation of bone microdamage has been proposed as one factor that contributes to increased skeletal fragility with age and that may increase the risk for fracture in older women. This paper reviews the current status and understanding of microdamage physiology and its importance to skeletal fragility. Several questions are addressed: Does microdamage exist in vivo in bone? If it does, does it impair bone quality? Does microdamage accumulate with age, and is the accumulation of damage with age sufficient to cause a fracture? The nature of the damage repair mechanism is reviewed, and it is proposed that osteoporotic fracture may be a consequence of a positive feedback between damage accumulation and the increased remodeling space associated with repair.

The response of rat tibiae to incremental bouts of mechanical loading: a quantum concept for bone formation.

To investigate the minimum number of loading bouts necessary to produce new lamellar or woven bone formation, and the time required for its initiation, bone formation was measured in 32 retired breeder female Sprague-Dawley rats following one, two, three, or five bouts of applied loading. Bending forces of 54 N were applied to right tibiae using a four-point loading apparatus, and left tibiae served as contralateral controls. Loading was applied as a sine wave with a frequency of 2 Hz for 18 s (36 cycles) per loading bout. Rats were injected with alizarin on day 1 and calcein on days 5 and 12, and were killed on day 19. One bout of loading was sufficient to increase the periosteal woven bone surface (Wb.Pm/B.Pm) from 0% to 40% (p < 0.01), and to 80% after five bouts of loading (p < 0.01), with a dose-response relationship for increases in Wb.Pm/B.Pm (p < 0.0001), mineral apposition rate (Wb.AR; p = 0.002), and bone formation rate (Wb.BFR/BS; p = 0.0001). In the first labeling period (days 1-5), the endocortical lamellar bone forming surface (BSf/BS) was increased slightly (p < 0.05), but no significant differences were shown for BFR/BS or MAR. From days 5 to 19, right tibiae showed a dose-response increase in BFR/BS (p = 0.002) and BSf/BS (p = 0.008), but not MAR. These results are consistent with a "quantum" model of bone formation such that a "quantum" of bone cells is activated in response to the loading bout and the strain magnitude dictate the size or microstructural organization of a given packet of new bone. Conversely, the distributed nature of loading may define the recruitment, rather than size, of new packets of bone.

Skeletal adaptations to mechanical usage: results from tibial loading studies in rats.

Wolff's law defines a static relationship between stress trajectories and trabecular architecture. More recent theories have attempted to describe the dynamic relationship between the form of bone and its mechanical environment. Frost's mechanostat theory is unique among these in its distinction between modeling and remodeling processes, lamellar and woven bone formation, mechanical usage windows for activation and its application to disorders of bone and mineral metabolism. Our studies suggest that lamellar and woven bone formation are very different not only in histological appearance, but in the temporal characteristics of their formation. Thus, it is important to distinguish these two histological types when interpreting studies of adaptive bone formation. Studies using the in vivo 4-point bending model in rat tibiae show that static loads do not play a role in mechanotransduction and that bone formation is threshold-driven and dependent on strain rate, amplitude and duration of loading. They have also provided strong indirect evidence that mechanical strains cause interstitial fluid flow that, in turn, activates the bone cell response. Based on these observations, we hypothesize that strain rate determines the vigor of osteoblastic activity and the regularity of loading bouts determines osteoblast recruitment in a "quantum" fashion.

Cancellous bone microdamage in the proximal femur: influence of age and osteoarthritis on damage morphology and regional distribution.

This study describes the in vivo distribution of cancellous bone microdamage in the proximal femur of an autopsy control sample. In addition, in vivo microdamage in the region medial to the greater trochanter of the proximal femur is compared between patients with severe osteoarthritis and controls. Taken at autopsy, the control group comprised normal right proximal femora that were then cut in the coronal plane with an Exakt saw (n = 12; aged 20-83 years). Cancellous bone samples were taken from the subchondral principal compressive region, the medial principal compressive region, and medial to the greater trochanter. A cancellous bone core biopsy was taken of the region medial to the greater trochanter (of the proximal femur) from patients with primary osteoarthritis undergoing total hip replacement surgery (n = 33; aged 37-85 years). Samples were embedded in resin, and in vivo microdamage identified in 70-microm-thick sections using the basic fuchsin en bloc staining technique. Microdamage was similar in all proximal femur sites in controls, except in the subchondral principal compressive region, where a significantly smaller crack length (microm) was identified (p < 0.05). In the region medial to the greater trochanter, osteoarthritic vs. control group comparisons showed that the crack density (#/mm(2)) and crack surface density (mm/mm(2)) were not significantly different, but crack length was significantly less (p < 0.03) and damage volume fraction was significantly increased for osteoarthritics (p < 0.005). The osteoarthritic and control data for crack density, and the osteoarthritic data for damage volume fraction, showed a nonlinear increase with age. Furthermore, crack length was not dependent on damage volume fraction or age for either the osteoarthritic or control group. This study identified differences in microdamage between osteoarthritic and autopsy control cases. We hypothesize that these results are consistent with the reported bone material property differences for osteoarthritis. In addition, the relatively uniform distribution of microdamage in the control group suggests that the principal components of the femoral cancellous bone network are equally exposed to deformations resulting in microdamage. Further study into the factors that influence the accumulation and skeletal distribution of microdamage is fundamental to understanding skeletal health.

Comparisons between bone and cementum compositions and the possible basis for their layered appearances.

In humans, age estimation from the adult skeleton represents an attempt to determine chronological age based on growth and maturational events. In teeth, such events can be characterized by appositional growth layers in midroot cementum. The purpose of this study was to determine the underlying cause of the layered microstructure of human midroot cementum. Whether cementum growth layers are caused by changes in relative mineralization, collagen packing and/or orientation, or by variations in organic matrix apposition was investigated by subjecting midroot sections of human canine teeth to analysis using polarized light and scanning electron microscopy (SEM). Polarized light was used to examine transverse midroot sections in both mineralized and demineralized states. Mineralized sections were also reexamined following subsequent decollagenization. Polarized light was additionally used in the examination of mineralized sections taken transversely, longitudinally, and obliquely from the same tooth root. From the birefringence patterns it was concluded that collagen orientation does not change with varying section plane. Instead, the mineral phase was most responsible for the birefringence of the cementum. SEM studies suggested that neither collagen packing nor collagen orientation change across the width of the cementum, confirming and validating the results of the polarized light examination. Also, SEM analysis using electron backscatter and the electron probe suggested no changes in the mean atomic number density, calcium, phosphate, and sulfur levels across the width of the cementum. Therefore, we conclude that crystalline orientation and/or size is responsible for the layered appearance of cementum.

Does suppression of bone turnover impair mechanical properties by allowing microdamage accumulation?

One plausible purpose of bone turnover is to repair bone microdamage. We hypothesized that suppression of bone turnover impairs bone quality by allowing accumulation of microdamage. We investigated the effect of high-dose etidronate (EHDP) on bone's mechanical properties and microdamage accumulation. Skeletally mature beagles, 1-2 years old at the beginning of the study, were treated with daily injections of vehicle or EHDP at 0.5 mg/kg per day or 5.0 mg/kg per day for 1 year. X-rays were taken at baseline and monthly from 7 to 12 months. Bones were taken upon sacrifice and biomechanical tests, histomorphometry, and microdamage analyses were performed. Fractures of ribs and/or thoracic spinous processes were found in 10 of 11 dogs treated with the higher dose EHDP. Only one fracture of a thoracic spinous process was found in dogs treated with the lower dose EHDP, and no fractures were found in the vehicle controls. Biomechanical tests showed reduced mechanical strength in ribs and lumbar vertebrae, but not in the femoral diaphysis or thoracic spinous process in the higher dose EHDP group. Histomorphometric measurements showed a significant reduction of cancellous bone turnover in both EHDP-treated groups compared with controls. In dogs treated with the higher dose EHDP, activation frequency was reduced to zero in both cortical and cancellous bone. Osteoid volume increased significantly, especially in trabecular bone, resulting in reduced mineralized bone volume in the higher dose EHDP group. Microcrack numerical density (Cr.Dn) increased significantly only in the lumbar vertebral body in the higher dose EHDP group, but not in the rib or thoracic spinous process where fractures occurred. These findings show that suppression of bone turnover using high doses of EHDP is associated with fractures of the ribs and spinous processes in dogs. This is most likely the result of excessive amounts of unmineralized bone produced by the inhibition of mineralization at these high doses, rather than by the accumulation of microdamage.

Prostaglandin E2 enhances alveolar bone formation in the rat mandible.

Prostaglandin E2 (PGE2) induces bone formation in stress-bearing bones. The mandible, a stress-bearing bone, is loaded daily during mastication. The aim of this study was to determine if PGE2 delivered locally to the mandible over 20 days enhances alveolar bone deposition. In 18 Lewis rats, controlled-release pellets containing PGE2 were implanted on the buccal aspect on the left-hand side of the mandible, mesial to the root of the first molar. Controlled-release pellets locally delivered 0.1, 0.05, or 0.025 mg/day of PGE2. The right side of the mandible was used as a matched control for each animal. Six sham-treated animals were implanted with a placebo pellet. On days 7 and 19, animals were injected with the bone markers tetracycline and calcein, respectively. On day 21, animals were sacrificed and undecalcified tissues obtained for morphometrical analysis. Morphometrical measurements were analyzed by paired t test to determine differences between the matched samples and one-way ANOVA to compare the different treatment groups. A significant increase in alveolar bone area was observed in mandibles treated with 0.1 and 0.05 mg/day when compared with matched controls and the placebo group. This was accompanied by a significant increase in alveolar bone height and width. The proportions of double-labeled surface (dLS), the mineral apposition rate (MAR), and bone formation rate (BFR) were significantly increased in mandibles treated with the two higher doses of PGE2. The proportion of resorptive surface (RS) was significantly reduced in these two groups. It is concluded that PGE2 induces alveolar bone formation in the mandible when locally delivered at a dose of 0.1 or 0.05 mg/day for 20 days.

Effects of suppressed bone turnover by bisphosphonates on microdamage accumulation and biomechanical properties in clinically relevant skeletal sites in beagles.

We recently demonstrated that suppression of bone remodeling allows microdamage to accumulate, leading to reduced bone toughness in the rib cortex of dogs. This study evaluates the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties at clinically relevant skeletal sites in the same dogs. Thirty-six female beagles, 1-2 years old, were divided into three groups. The control group was treated daily for 12 months with saline vehicle (CNT). The remaining two groups were treated daily with risedronate at a dose of 0.5 mg/kg per day (RIS), or alendronate at 1.0 mg/kg per day (ALN) orally. The doses of these bisphosphonates were six times the clinical doses approved for treatment of osteoporosis in humans. After killing, the L-1 vertebra was scanned by dual-energy X-ray absorptiometry (DXA), and the L-2 vertebra and right ilium were assigned to histomorphometry. The L-3 vertebra, left ilium, Th-2 spinous process, and right femoral neck were used for microdamage analysis. The L-4 vertebra and Th-1 spinous process were mechanically tested to failure in compression and shear, respectively. One year treatment with risedronate or alendronate significantly suppressed trabecular remodeling in vertebrae (RIS 90%, ALN 95%) and ilium (RIS 76%, ALN 90%) without impairment of mineralization, and significantly increased microdamage accumulation in all skeletal sites measured. Trabecular bone volume and vertebral strength increased significantly following 12 month treatment. However, normalized toughness of the L-4 vertebra was reduced by 21% in both RIS (p = 0.06) and ALN (p = 0.05) groups. When the two bisphosphonate groups were pooled in a post hoc fashion for analysis, this reduction in toughness reached statistical significance (p = 0.02). This study demonstrates that suppression of trabecular bone turnover by high doses of bisphosphonates is associated with increased vertebral strength, even though there is significant microdamage accumulation and a reduction in the intrinsic energy absorption capacity of trabecular bone.

Assessment of cancellous bone quality in severe osteoarthrosis: bone mineral density, mechanics, and microdamage.

The role of bone microdamage (microscopic cracks or microcracks and ultrastructural collagen matrix and bone mineral damage) in diseases such as osteoarthrosis and osteoporosis is poorly understood. Microdamage accumulation in vivo is influenced by age and cyclic loading, therefore, it would be useful if the burden of microdamage in bone could be assessed by noninvasive measures such as the radiological measurement of bone mineral density (BMD). The aim of this study was to investigate the relationship between BMD, compressive strength and stiffness, and microdamage in the cancellous bone of the proximal femur in patients with severe osteoarthrosis. Trabecular bone core samples, from the intertrochanteric region of the femur, were obtained from 34 patients, with a mean age of 70.3 +/- 11.1 years, undergoing total hip arthroplasty for osteoarthrosis. Cores selected from contact X-ray images were used for BMD measurement, compressive mechanical testing or left untested (uncrushed), en bloc staining for microdamage, and bone histomorphometry. The study shows a strong dependence of both the elastic modulus and ultimate failure stress of the bone samples on BMD and a significant relationship between the elastic modulus and trabecular anisotropy (Tr. An). In multiple linear regression, BMD and Tr. An together account for about 70% of the variance in the elastic modulus. Then including microcrack crack density (Cr.Dn) and damage volume fraction (DxV/BV) variables, Tr. An alone accounts for a relatively small amount of the variation (8.5%) in ultimate failure stress and elastic modulus. The Cr.Dn accounts for more of the variation in the ultimate failure stress than in the elastic modulus (50% vs. 7%). In this experiment, data for Cr.Dn provide a measure of damage associated with the ultimate failure of cancellous bone. In specimens that were not mechanically tested, in vivo microcrack accumulation increases exponentially with age. In conclusion, data from this study suggest that BMD and Cr.Dn are the major determinants of cancellous bone strength, whereas BMD and Tr. An are major determinants of cancellous bone stiffness. In bone specimens subjected to compressive testing there was no relationship between microdamage and BMD, suggesting that BMD cannot be used to monitor changes in the mechanical properties of bone due to microdamage accumulation.

Three-dimensional confocal images of microdamage in cancellous bone.

The accumulation of microdamage in bone may contribute to loss of bone quality in osteoporosis, and the role of microdamage in the etiology of fatigue fractures is unknown. Microdamage created during testing, ex vivo, can increase the fragility of bone by decreasing the load necessary to cause fracture. Microdamage can also accumulate in vivo, but its influence on bone fragility is unknown. To date, stained microcracks are the only criteria to have been correlated with bone mechanics, leaving the influence of ultrastructural damage on bone fragility open for scrutiny. Staining en bloc has identified three morphological features in the tissue, discrete microcracks, cross-hatch staining, and diffuse staining. The relationship between these features and their identification as microdamage remains equivocal. The purpose of this study was to investigate the three-dimensional nature of microdamage in cancellous bone and also to describe stained microcracks, cross-hatch staining, and diffuse staining and to determine whether they all relate to microdamage in bone. Laser scanning confocal microscopy that provides improved spatial resolution over bright-field microscopy was used to visualize bone damage. It was found that crack surface density was highly correlated with crack density (r = 0.95, p < 0.0001), suggesting that the crack surface of preexisting cracks increases as new cracks are formed or submicroscopic cracks become visible under bright-field microscopy. Cross-hatch staining and diffuse staining included ultra-microcracks about 10 microm in length. The ultra-microcracks in cross-hatch staining were organized in bands and surrounded by diffuse staining. This study demonstrates that damage in bone occurs over a wide range and that discrete microcracks, cross-hatch staining, and diffuse staining are all indicative of bone damage. The diffuse staining still evident in association with the ultra-microcracks seen in cross-hatch staining and diffuse staining is probably due to damage at a still smaller scale than we have been able to investigate.