CAMKK2 is upregulated in primary human osteoarthritis and its inhibition protects against chondrocyte apoptosis.

OBJECTIVE: To investigate the role of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) in human osteoarthritis. MATERIALS AND METHODS: Paired osteochondral plugs and articular chondrocytes were isolated from the relatively healthier (intact) and damaged portions of human femoral heads collected from patients undergoing total hip arthroplasty for primary osteoarthritis (OA). Cartilage from femoral plugs were either flash frozen for gene expression analysis or histology and immunohistochemistry. Chondrocyte apoptosis in the presence or absence of CAMKK2 inhibition was measured using flow cytometry. CAMKK2 overexpression and knockdown in articular chondrocytes were achieved via Lentivirus- and siRNA-mediated approaches respectively, and their effect on pro-apoptotic and cartilage catabolic mechanisms was assessed by immunoblotting. RESULTS: CAMKK2 mRNA and protein levels were elevated in articular chondrocytes from human OA cartilage compared to paired healthier intact samples. This increase was associated with elevated catabolic marker matrix metalloproteinase 13 (MMP-13), and diminished anabolic markers aggrecan (ACAN) and type II collagen (COL2A1) levels. OA chondrocytes displayed enhanced apoptosis, which was suppressed following pharmacological inhibition of CAMKK2. Levels of MMP13, pSTAT3, and the pro-apoptotic marker BAX became elevated when CAMKK2, but not its kinase-defective mutant was overexpressed, whereas knockdown of the kinase decreased the levels of these proteins. CONCLUSIONS: CAMKK2 is upregulated in human OA cartilage and is associated with elevated levels of pro-apoptotic and catabolic proteins. Inhibition or knockdown of CAMKK2 led to decreased chondrocyte apoptosis and catabolic protein levels, whereas its overexpression elevated them. CAMKK2 may be a therapeutic target to prevent or mitigate human OA.

Systemic inhibition or global deletion of CaMKK2 protects against post-traumatic osteoarthritis.

OBJECTIVE: To investigate the role of Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2) in post-traumatic osteoarthritis (PTOA). METHODS: Destabilization of the medial meniscus (DMM) or sham surgeries were performed on 10-week-old male wild-type (WT) and Camkk2-/- mice. Half of the DMM-WT mice and all other cohorts (n = 6/group) received tri-weekly intraperitoneal (i.p.) injections of saline whereas the remaining DMM-WT mice (n = 6/group) received i.p. injections of the CaMKK2 inhibitor STO-609 (0.033 mg/kg body weight) thrice a week. Study was terminated at 8- or 12-weeks post-surgery, and knee joints processed for microcomputed tomography imaging followed by histology and immunohistochemistry. Primary articular chondrocytes were isolated from knee joints of 4-6-day-old WT and Camkk2-/- mice, and treated with 10 ng/ml interleukin-1ß (IL)-1ß for 24 or 48 h to investigate gene and protein expression. RESULTS: CaMKK2 levels and activity became elevated in articular chondrocytes following IL-1ß treatment or DMM surgery. Inhibition or absence of CaMKK2 protected against DMM-associated destruction of the cartilage, subchondral bone alterations and synovial inflammation. When challenged with IL-1ß, chondrocytes lacking CaMKK2 displayed attenuated inflammation, cartilage catabolism, and resistance to suppression of matrix synthesis. IL-1ß-treated CaMKK2-null chondrocytes displayed decreased IL-6 production, activation of signal transducer and activator of transcription 3 (Stat3) and matrix metalloproteinase 13 (MMP13), indicating a potential mechanism for the regulation of inflammatory responses in chondrocytes by CaMKK2. CONCLUSIONS: Our findings reveal a novel function for CaMKK2 in chondrocytes and highlight the potential for its inhibition as an innovative therapeutic strategy in the prevention of PTOA.

Three years of alendronate treatment does not continue to decrease microstructural stresses and strains associated with trabecular microdamage initiation beyond those at 1 year.

UNLABELLED: The effects of a 3-year alendronate treatment on trabecular stresses/strains associated with microdamage initiation were investigated using finite element modeling (FEM). Severely damaged trabeculae in the low-dose treatment group were associated with increased stresses compared with the high-dose treatment group (p = 0.006) and approached significance in the control group (p = 0.02). INTRODUCTION: Alendronate, a commonly prescribed anti-remodeling agent, decreases fracture risk in the vertebrae, hip, and wrist of osteoporotic individuals. However, evaluation of microdamage accumulation in animal and human studies shows increased microdamage density relative to controls. Microstructural von Mises stresses associated with severe and linear damage have been found to decrease after 1 year of alendronate treatment. In the present study, stresses/strains associated with damage were assessed after 3 years of treatment to determine whether they continued to decrease with increased treatment duration. METHODS: Microdamaged trabeculae visualized with fluorescent microscopy were associated with stresses and strains obtained using image-based FEM. Stresses/strains associated with severe, diffuse, and linearly damaged and undamaged trabeculae were compared among groups treated for 3 years with an osteoporotic treatment dose of alendronate, a Paget's disease treatment dose of alendronate, or saline control. Architectural characteristics and mineralization were also analyzed from three-dimensional microcomputed tomography reconstructed images. RESULTS: Severely damaged trabeculae in the osteoporotic treatment dose group were associated with increased stress compared with the Paget's disease treatment dose group (p = 0.006) and approached significance compared to the control group (p = 0.02). Trabecular mineralization in severely damaged trabeculae of the low-dose treatment group was significantly greater compared to severely damaged trabeculae in the high-dose treatment and control group, suggesting that changes at the tissue level may play a role in these findings. CONCLUSIONS: Trabecular level stresses associated with microdamage do not continue to decrease with prolonged alendronate treatment. Changes in mineralization may account for these findings.

Bone quality in an ovariectomized monkey animal model treated with two doses of teriparatide for either 18 months, or 12 months followed by withdrawal for 6 months.

Previous studies of ovariectomized (OVX) monkeys, treated with recombinant human parathyroid hormone (PTH) (1-34) at 1 or 5 µg/kg/day for 18 months or for 12 months followed by 6 months withdrawal from treatment, displayed significant changes in geometry, histomorphometry, and bone quality, but without strict tissue age criteria, of the midshaft humerus. Since bone quality significantly depends on tissue age among other factors, the aim of the present study was to establish the bone-turnover independent effects of two doses of PTH, as well as the effects of treatment withdrawal on bone quality by measuring bone material composition at precisely known tissue ages ranging from osteoid, to mineralized tissue older than 373 days. Raman microspectroscopic analysis of bone tissue from the mid-shaft humerus of OVX monkeys demonstrated that the clinically relevant dose of PTH administered for 18 months reverses the effects of ovariectomy on bone quality when compared against SHAM. Both doses investigated in this study restore the mineralization regulation mechanisms to SHAM levels. The study also showed that the beneficial effects induced by 12 months of clinically relevant PTH therapy were sustained after six months of therapy withdrawal.

Compromised osseous healing of dental extraction sites in zoledronic acid-treated dogs.

UNLABELLED: The goal of this study was to document how treatment with high doses of zoledronic acid affects dental extraction healing. Our results, showing significantly compromised osseous healing within the socket as well as presence of exposed bone and development of a sequestrum in one animal, provide a building block toward understanding osteonecrosis of the jaw. PURPOSE: The goal of this study was to document how treatment with a bisphosphonate affects the bone tissue following dental extraction. METHODS: Skeletally mature female beagle dogs were either untreated controls (CON) or treated with intravenous zoledronic acid (ZOL). Following the extraction of the fourth premolars, healing was allowed for 4 or 8 weeks. Properties of the extraction site were assessed using microcomputed tomography (micro-CT) and dynamic histomorphometry. RESULTS: The initial infilling of the extraction socket with bone was not affected by ZOL, but subsequent removal of this bone was significantly suppressed compared to CON. After 8 weeks of healing, the alveolar cortical bone adjacent to the extraction socket had a remodeling rate of ∼50% per year in CON animals while ZOL-treated animals had a rate of <1% per year. One ZOL-treated animal developed exposed bone post-extraction which eventually led to the formation of a sequestrum. Assessment of the sequestrum with micro-CT and histology showed that it had features consistent with those reported in humans with osteonecrosis of the jaw. CONCLUSIONS: These results, showing significantly compromised post-extraction osseous healing as well as presence of exposed bone and development of a sequestrum in one ZOL animal, provide a building block toward understanding the pathophysiology of osteonecrosis of the jaw.

Why bones bend but don't break.

The musculoskeletal system is adept at dissipating potentially damaging energy that could accelerate fracture consequent to multiple loading cycles. Microstructural damage reduces bone's residual properties, but prevents high stresses within the material by dissipating energy that can lead to eventual failure. Thus skeletal microdamage can be viewed as an adaptive process to prevent bone failure by dissipating energy. Because a damaged bone has reduced strength and stiffness, it must be repaired, so bone has evolved a system of self-repair that relies on microdamage-stimulated signaling mechanisms. When repair cannot occur quickly enough, low energy stress fractures can occur. The regulating effects of muscle also prevent failure by controlling where high stresses occur. Acting synergistically, muscle forces dissipate energy by appropriately regulating accelerations and decelerations of the limbs during movement. When muscles become fatigued, these functions are constrained, larger amounts of energy are imparted to bone, increasing the likelihood of microstructural damage and fracture. Thus, healthy bones are maintained by the ability of the musculoskeletal system to dissipate the energy through synergistic muscular activity and through the maintenance of microstructural and material properties that allow for crack initiation, but also for their repair.

Histomorphometric changes by teriparatide in alendronate-pretreated women with osteoporosis.

SUMMARY: The level of increased bone formation after 24 months of treatment with teriparatide (rhPTH (1-34), TPTD) is similar in patients who were either treatment-naïve (TN) or had lower bone turnover initially due to previous alendronate (ALN) therapy. INTRODUCTION: Bone anabolic effects of TPTD in postmenopausal women with osteoporosis may be blunted during the initial phase after switching from ALN to TPTD. To explore the long-term implications, we examined histomorphometric and biochemical markers of bone turnover of patients on TPTD therapy after long-term ALN treatment. METHODS: Paired biopsies were obtained after tetracycline double labeling at baseline and after 24 months of TPTD treatment from 29 ALN-pretreated (64.5 ± 16.4 months) and 16 TN patients. Biochemical markers were measured at baseline, during the treatment, or at study end. RESULTS: Compared with the baseline, after 24-month TPTD, activation frequency (Ac.F.) and osteoid surface (OS) increased in both groups: 0.11-0.34 cycles per year, 3.96-9.8% in the ALN-pretreated group and 0.19-0.33 cycles per year, 6.2-11.3% (p < 0.05) in the TN group, respectively. Biochemical and histomorphometric markers correlated positively both at baseline and endpoint. Serum amino terminal propeptide of type I procollagen (PINP) correlated with Ac.F. (r = 0.57, p < 0.001 and r = 0.48, p < 0.01) and OS (r = 0.51, p < 0.01 and r = 0.56, p < 0.01) at baseline and endpoint, respectively. Following 3 months of treatment, increases in biochemical markers like PINP predicted the increase in Ac.F. (r = 0.52, p < 0.01) and OS (r = 0.54, p < 0.01) after 24 months. CONCLUSIONS: The increased level of formation is similar in patients who were either TN or had lower bone turnover initially due to previous ALN therapy. Elevated bone formation in postmenopausal women with osteoporosis was sustained over a 24-month period by TPTD. Biochemical markers of bone formation are a good surrogate for the assessment of TPTD effects.

Ovariectomy stimulates and bisphosphonates inhibit intracortical remodeling in the mouse mandible.

OBJECTIVE: The pathophysiology of osteonecrosis of the jaw (ONJ) is thought to be linked to suppression of intracortical remodeling. The aim of this study was to determine whether mice, which normally do not undergo appreciable amounts of intracortical remodeling, could be stimulated by ovariectomy to remodel within the cortex of the mandible and if bisphosphonates (BPs) would suppress this intracortical remodeling. MATERIAL AND METHODS: Skeletally mature female C3H mice were either ovariectomized (OVX) or SHAM operated and treated with two intravenous doses of zoledronic acid (ZOL, 0.06 mg/kg body weight) or vehicle (VEH). This ZOL dose corresponds to the dose given to patients with cancer on a mg/kg basis, adjusted for body weight. Calcein was administered prior to sacrifice to label active formation sites. Dynamic histomorphometry of the mandible and femur was performed. RESULTS: Vehicle-treated OVX animals had significantly higher (eightfold) intracortical remodeling of the alveolar portion of the mandible compared to sham--this was significantly suppressed by ZOL treatment. At all skeletal sites, overall bone formation rate was lower with ZOL treatment compared to the corresponding VEH group. CONCLUSIONS: Under normal conditions, the level of intracortical remodeling in the mouse mandible is minimal but in C3H mice it can be stimulated to appreciable levels with ovariectomy. Based on this, if the suppression of intracortical remodeling is found to be part of the pathophysiology of ONJ, the ovariectomized C3H mouse could serve as a useful tool for studying this condition.

Alendronate treatment results in similar levels of trabecular bone remodeling in the femoral neck and vertebra.

SUMMARY: We aimed to determine whether trabecular bone in sites that have different surface-based remodeling rates, the femoral neck and vertebra, are differently affected by alendronate treatment. Alendronate treatment resulted in similar levels of turnover in both sites, suggesting that a lower limit of bone turnover suppression with alendronate may exist. INTRODUCTION: Bone turnover suppression in sites that already have a low surface-based remodeling rate may lead to oversuppression that could have negative effects on the biomechanical properties of bone. The goal was to determine how alendronate suppresses bone turnover at sites with different surface-based remodeling rates. METHODS: Dynamic histomorphometric parameters were assessed in trabecular bone of the femoral neck and lumbar vertebrae obtained from skeletally mature beagles treated with saline (1 ml/kg/day) or alendronate (ALN 0.2 or 1.0 mg/kg/day). The ALN0.2 and ALN1.0 doses approximate, on a milligram per kilogram basis, the clinical doses used for the treatment of postmenopausal osteoporosis and Paget's disease, respectively. RESULTS: Alendronate treatment resulted in similar absolute levels of bone turnover in the femoral neck and vertebrae, although the femoral neck had 33% lower pre-treatment surface-based remodeling rate than the vertebra (p < 0.05). Additionally, the high dose of alendronate (ALN 1.0) suppressed bone turnover to similar absolute levels as the low dose of alendronate (ALN 0.2) in both sites. CONCLUSIONS: Alendronate treatment may result in a lower limit of trabecular bone turnover suppression, suggesting that sites of low pre-treatment remodeling rate are not more susceptible to oversuppression than those of high pre-treatment remodeling rate.

Changes in non-enzymatic glycation and its association with altered mechanical properties following 1-year treatment with risedronate or alendronate.

SUMMARY: One year of high-dose bisphosphonate (BPs) therapy in dogs allowed the increased accumulation of advanced glycation end-products (AGEs) and reduced postyield work-to-fracture of the cortical bone matrix. The increased accumulation of AGEs in these tissues may help explain altered bone matrix quality due to the administration of BPs in animal models INTRODUCTION: Non-enzymatic glycation (NEG) is a posttranslational modification of the organic matrix that results in the formation of advanced glycation end-products (AGEs). In bone, the accumulation of AGEs play an important role in determining fracture resistance, and elevated levels of AGEs have been shown to adversely affect the bone's propensity to brittle fracture. It was thus hypothesized that the suppression of tissue turnover in cortical bone due to the administration of bisphosphonates would cause increased accumulation of AGEs and result in a more brittle bone matrix. METHODS: Using a canine animal model (n = 12), we administered daily doses of a saline vehicle (VEH), alendronate (ALN 0.20, 1.00 mg/kg) or risedronate (RIS 0.10, 0.50 mg/kg). After a 1-year treatment, the mechanical properties, intracortical bone turnover, and the degree of nonenzymatic cross-linking of the organic matrix were measured from the tibial cortical bone tissue of these animals. RESULTS: There was a significant accumulation of AGEs at high treatment doses (+49 to + 86%; p < 0.001), but not at doses equivalent to those used for the treatment of postmenopausal osteoporosis, compared to vehicle. Likewise, postyield work-to-fracture of the tissue was significantly reduced at these high doses (-28% to -51%; p < 0.001) compared to VEH. AGE accumulation inversely correlated with postyield work-to-fracture (r (2) = 0.45; p < 0.001), suggesting that increased AGEs may contribute to a more brittle bone matrix. CONCLUSION: High doses of bisphosphonates result in the accumulation of AGEs and a reduction in energy absorption of cortical bone. The increased accumulation of AGEs in these tissues may help explain altered bone matrix quality due to the administration of BPs in animal models.

Mandibular necrosis in beagle dogs treated with bisphosphonates.

OBJECTIVES - To test the effect of bisphosphonate (BP) treatment for up to 3 years on bone necrosis and osteocyte death in the mandible using a canine model. MATERIALS AND METHODS - Dogs were treated with clinical doses of oral alendronate (ALN, 0.2 or 1.0 mg/kg/day) for 1 or 3 years. In a separate study, dogs were treated with i.v. zoledronate (ZOL) at 0.06 mg/kg/day for 6 months. En bloc staining was used to identify necrotic areas in the mandible; viable osteocytes were identified using lactate dehydrogenase. RESULTS - None of the treatments was associated with exposed bone, but 17-25% of dogs treated for 1 year and 25-33% of dogs treated for 3 years with ALN showed pockets of dead bone. Necrotic areas had no viable osteocytes and were void of patent canaliculi. No control animals demonstrated necrotic bone. ZOL treatment for 6 months was associated with osteocyte death greater than that seen in animals treated with ALN or saline. It is not clear whether osteocyte death occurs because of direct toxic effects of BPs, or because suppressed remodelling fails to renew areas that naturally undergo cell death. Necrotic areas are also associated with bone other than the mandible, e.g. the rib, which normally undergo high rates of remodelling. CONCLUSIONS - Reduced remodelling rate using BPs may contribute to the pathogenesis of bone matrix necrosis. The development of an animal model that mimics important aspects of BP-related osteonecrosis of the jaw is important to understanding the pathogenesis of osteonecrosis.

The effect of the microscopic and nanoscale structure on bone fragility.

Bone mineral density is the gold-standard for assessing bone quantity and diagnosing osteoporosis. Although bone mineral density measurements assess the quantity of bone, the quality of the tissue is an important predictor of fragility. Understanding the macro- and nanoscale properties of bone is critical to understanding bone fragility in osteoporosis. Osteoporosis is a disease that affects more than 75 million people worldwide. The gold standard for osteoporosis prognosis, bone mineral density, primarily measures the quantity of bone in the skeleton, overlooking more subtle aspects of bone's properties. Bone quality, a measure of bone's architecture, geometry and material properties, is evaluated via mechanical, structural and chemical testing. Although decreased BMD indicates tissue fragility at the clinical level, changes in the substructure of bone can help indicate how bone quality is altered in osteoporosis. Additionally, mechanical properties which can quantify fragility, or bone's inability to resist fracture, can be changed due to alterations in bone architecture and composition. Recent studies have focused on examination of bone on the nanoscale, suggesting the importance of understanding the interactions of the mineral crystals and collagen fibrils and how they can alter bone quality. It is therefore important to understand alterations in bone that occur at the macro-, micro- and nanoscopic levels to determine what parameters contribute to decreased bone quality in diseased tissue.

Strontium ranelate does not stimulate bone formation in ovariectomized rats.

INTRODUCTION: Strontium ranelate (SrR) is suggested to function as a dual-acting agent in the treatment of postmenopausal osteoporosis with anti-resorptive and anabolic skeletal benefits. We evaluated the effects of SrR on the skeleton in ovariectomized (OVX) rats and evaluated the influence of dietary calcium. METHODS: Three-month old virgin female rats underwent ovariectomy (OVX, n = 50) or SHAM surgery (SHAM, n = 10). Four weeks post-surgery, rats were treated daily by oral gavage with distilled water (10 ml/kg/day) or SrR (25 or 150 mg/kg/day) for 90 days. Separate groups of animals for each dose of SrR were fed a low (0.1%) or normal (1.19%) calcium (Ca) diet. Static and dynamic histomorphometry, DXA, mu-CT, mechanical testing, and serum and skeletal concentrations of strontium were assessed. RESULTS: SrR at doses of 25 and 150 mg/kg/day did not increase bone formation on trabecular or periosteal bone surfaces, and failed to inhibit bone resorption of trabecular bone regardless of Ca intake. There were no improvements in bone mass, volume or strength with either dose of SrR given normal Ca. CONCLUSION: These findings demonstrate that SrR at dosages of 25 and 150 mg/kg/day did not stimulate an anabolic bone response, and failed to improve the bone biomechanical properties of OVX rats.

Proprioception, gait kinematics, and rate of loading during walking: are they related?

The cyclic nature of walking can lead to repetitive stress and associated complications due to the rate of loading (ROL) experienced by the body at the initial contact of the foot with the ground. An individual's gait kinematics at initial contact has been suggested to give rise to the ROL, and a repetitive, high ROL may lead to several disorders, including osteoarthritis. Additionally, proprioception, the feedback signaling of limb position and movement, may play a role in how the foot strikes the ground and thus, the ROL. Our goal was to explore the relationship between proprioception, gait kinematics and ROL. Thirty-eight women were recruited for gait analysis, and the gait characteristics 50 ms prior to and at initial contact were examined. Two proprioception tests, joint angle reproduction and threshold to detect passive motion were used to examine the subject's proprioceptive acuity. Our results indicate that individuals with a larger knee angle (i.e., greater extension) 50 ms prior to initial contact (IC) experience a higher ROL during gait and have poorer proprioceptive scores. However, it remains unclear whether poor proprioception causes a high ROL or if a high ROL damages the mechanoreceptors involved in proprioception, but the apparent relationship is significant and warrants further investigation.

Mineral anisotropy in mineralized tissues is similar among species and mineral growth occurs independently of collagen orientation in rats: results from acoustic velocity measurements.

It has been reported that the mineral crystals in long bones have their c-axis aligned with the bone axis, presumably because collagen fibrils in bone also align with the bone axis. However, the predominant collagen orientation in bone often does not appear to be aligned with the mineral crystals, especially in rat primary bone. We hypothesized that mineral orientation in bone is not necessarily related to collagen orientation. An acoustic microscope was used to measure elastic constants of mineralized tissues from rat, cow, monkey, and human bone, and mineralized turkey leg tendon (MTLT). Measurements were made before and after demineralization with 10% ethylenediaminetetraacetic acid (EDTA) or decollagenization with 7% sodium hypochlorite. The elastic anisotropy ratio (AR) was defined as the ratio of the elastic coefficient in the longitudinal direction to the elastic coefficient in the transverse direction. Anisotropy ratios of mineralized tissues were not affected by formalin fixation or plastic embedding. An evaluation of tissues from the different species showed that the AR after decollagenization was not significantly different (p > 0.4, analysis of variance) among the groups, while AR after demineralization varied from 1.04 (rat bone) to 1.51 (MTLT). There was no correlation between AR after demineralization and AR after decollagenization (r = 0.13, p = 0.5). This showed that the elastic anisotropy of collagen is more variable than mineral anisotropy in bone and MTLT. Another experiment showed that mineralization of turkey leg tendon changes the elasticity of the collagen matrix, making it less anisotropic. A final, prospective experiment was performed in which tibiae of rats were subjected to mechanical loading for 16 weeks. After 12 days, new periosteal woven bone was observed on the tibiae and, after 16 weeks, this new bone was consolidated and mineralized. Mineral in the newly formed woven bone was virtually isotropic (AR = 1.07) after 12 days of loading, then became more anisotropic (AR = 1.52) after 16 weeks of mechanical loading, as the mineral density of the new bone increased. This increase in anisotropy of bone mineral occurred even though the collagen matrix was woven and had no measureable fibril orientation. We conclude that (1) collagen anisotropy and mineral anisotropy are not necessarily correlated in mineralized tissues, (2) mineralization can affect the collagen matrix elasticity of mineralized tissues, and (3) an organized mineral structure can form in the absence of an organized collagen matrix.

Partitioning a daily mechanical stimulus into discrete loading bouts improves the osteogenic response to loading.

A single 3-minute bout of mechanical loading increases bone formation in the rat tibia. We hypothesized that more frequent, shorter loading bouts would elicit a greater osteogenic response than a single 3-minute bout. The right tibias of 36 adult female Sprague-Dawley rats were subjected to 360 bending cycles per day of a 54 N force delivered in 1, 2, 4, or 6 bouts on each of the 3 loading days. Rats in the 6-bouts/day group received 60 bending cycles per bout (60 x 6); rats in the 4-bouts/day group received 90 bending cycles per bout (90 x 4); the 2- and 1-bouts/day groups received 180 and 360 bending cycles per bout, respectively (180 x 2 and 360 x 1). A nonloaded, age-matched control group (0 x 0) and two sham-bending groups (60 x 6 and 360 x 1) also were included. Fluorochrome labeling revealed a 10-fold increase in endocortical lamellar bone formation rate (BFR/bone surface [BS]) in the right tibia versus the left (nonloaded) side in the 60 x 6 bending group. Endocortical BFR/BS in the right tibia of the 4-, 2-, and 1-bout bending groups exhibited 8-, 4-, and 4-fold increases, respectively, over the control side. Relative (right minus left) values for endocortical BFR/BS, mineralizing surface (MS/BS), and mineral apposition rate (MAR) were 65-94% greater in the 90 x 4 and 60 x 6 bending groups compared to the 360 x 1 bending group. Sham-bending tibias exhibited relative endocortical bone formation values similar to those collected from the control (0 x 0) group. The data show that 360 daily loading cycles applied at intervals of 60 x 6 or 90 x 4 represent a more osteogenic stimulus than 360 cycles applied all at once, and that mechanical loading is more osteogenic when divided into discrete loading bouts. Presumably, bone cells become increasingly "deaf" to the mechanical stimulus as loading cycles persist uninterrupted, and by allowing a rest period between loading bouts, the osteogenic effectiveness of subsequent cycles can be increased.

Mechanical loading of diaphyseal bone in vivo: the strain threshold for an osteogenic response varies with location.

Bone tissue responds to elevated mechanical loading with increased bone formation, which is triggered either directly or indirectly by the mechanical strain engendered in the bone tissue. Previous studies have shown that mechanical strain magnitude must surpass a threshold before bone formation is initiated. The objective of this study was to estimate the strain thresholds at three different locations along the ulna of adult rats. We hypothesized that the strain threshold would be greater in regions of the ulna habitually subjected to larger mechanical strains. New bone formation was measured on the periosteal and endocortical surfaces of the ulnar diaphysis in adult female rats exposed to controlled dynamic loading. Axial, compressive loading was applied daily at five different magnitudes for a period of 2 weeks. Bone formation rate (BFR) was measured, using double-label histomorphometry at the ulnar middiaphysis and at locations 3 mm proximal and 3 mm distal to the middiaphysis. Loading induced lamellar bone formation on the periosteal surface that was greater at the distal ulnar location and lower at the proximal location when compared with the middiaphysis. Likewise, peak strains on the periosteal surface were greatest distally and less proximally. There was a significant dose-response relationship between peak strain magnitude and periosteal new bone formation when the mechanically induced strain surpassed a threshold. The strain threshold varied from 1343 microstrain (mu strain) proximally to 2284 mu strain at the midshaft to 3074 mu strain distally. Unlike the periosteal response to mechanical loading, there was not a clear dose-response relationship between applied load and bone formation on the endocortical surface. Endocortical strains were estimated to be < 20% of periosteal strains and may not have been sufficient to initiate a bone formation response. Our results show that the osteogenic response on the periosteal surface of the ulna depends on peak strain level once a strain threshold is surpassed. The threshold strain is largest distally, where locomotor bone strains are typically higher and smallest proximally where locomotor bone strains are lower.

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.