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.

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.

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.

Spontaneous perception of numerosity in pre-school children.

There is strong evidence that humans can make rough estimates of the numerosity of a set of items, almost from birth. However, as numerosity covaries with many non-numerical variables, the idea of a direct number sense has been challenged. Here we applied two different psychophysical paradigms to demonstrate the spontaneous perception of numerosity in a cohort of young pre-school children. The results of both tasks showed that even at that early developmental stage, humans spontaneously base the perceptual choice on numerosity, rather than on area or density. Precision in one of these tasks predicted mathematical abilities. The results reinforce strongly the idea of a primary number sense and provide further evidence linking mathematical skills to the sensory precision of the spontaneous number sense, rather than to mechanisms involved in handling explicit numerosity judgements or extensive exposure to mathematical teaching.

Estrogen depletion alters mineralization regulation mechanisms in an ovariectomized monkey animal model.

Ovariectomized animal models have been extensively used in osteoporosis research due to the resulting loss of bone mass. The purpose of the present study was to test the hypothesis that estrogen depletion alters mineralization regulation mechanisms in an ovariectomized monkey animal model. To achieve this we used Raman microspectroscopy to analyze humeri from monkeys that were either SHAM-operated or ovariectomized (N = 10 for each group). Measurements were made as a function of tissue age and cortical surface (periosteal, osteonal, endosteal) based on the presence of calcein fluorescent double labels. In the present work we focused on osteoid seams (defined as a surface with evident calcein labels, 1 µm distance away from the mineralizing front, and for which the Raman spectra showed the presence of organic matrix but not mineral), as well as the youngest mineralized tissue between the second fluorescent label and the mineralizing front, 1 µm inwards from the front with the phosphate mineral peak evident in the Raman spectra (TA1). The spectroscopically determined parameters of interest were the relative glycosaminoglycan (GAG) and pyridinoline (Pyd) contents in the osteoid, and the mineral content in TA1. At all three cortical surfaces, significant correlations were evident in the SHAM-operated animals between osteoid GAG (negative) and Pyd content, and mineral content, unlike the OVX animals. These results suggest that in addition to the well-established effects on turnover rates and bone mass, estrogen depletion alters the regulation of mineralization by GAGs and Pyd.

Sensory integration deficits support a dimensional view of psychosis and are not limited to schizophrenia.

Visual dysfunction is commonplace in schizophrenia and occurs alongside cognitive, psychotic and affective symptoms of the disorder. Psychophysical evidence suggests that this dysfunction results from impairments in the integration of low-level neural signals into complex cortical representations, which may also be associated with symptom formation. Despite the symptoms of schizophrenia occurring in a range of disorders, the integration deficit has not been tested in broader patient populations. Moreover, it remains unclear whether such deficits generalize across other sensory modalities. The present study assessed patients with a range of psychotic and nonpsychotic disorders and healthy controls on visual contrast detection, visual motion integration, auditory tone detection and auditory tone integration. The sample comprised a total of 249 participants (schizophrenia spectrum disorder n=98; bipolar affective disorder n=35; major depression n=31; other psychiatric conditions n=31; and healthy controls n=54), of whom 178 completed one or more visual task and 71 completed auditory tasks. Compared with healthy controls and nonpsychotic patients, psychotic patients trans-diagnostically were impaired on both visual and auditory integration, but unimpaired in simple visual or auditory detection. Impairment in visual motion integration was correlated with the severity of positive symptoms, and could not be accounted for by a reduction in processing speed, inattention or medication effects. Our results demonstrate that impaired sensory integration is not specific to schizophrenia, as has previously been assumed. Instead, sensory deficits are closely related to the presence of positive symptoms independent of diagnosis. The finding that equivalent integrative sensory processing is impaired in audition is consistent with hypotheses that propose a generalized deficit of neural integration in psychotic disorders.

Ovarian hormone depletion affects cortical bone quality differently on different skeletal envelopes.

The physical properties of bone tissue are determined by the organic and mineral matrix, and are one aspect of bone quality. As such, the properties of mineral and matrix are a major contributor to bone strength, independent of bone mass. Cortical bone quality may differ regionally on the three skeletal envelopes that compose it. Each of these envelopes may be affected differently by ovarian hormone depletion. Identifying how these regions vary in their tissue adaptive response to ovarian hormones can inform our understanding of how tissue quality contributes to overall bone strength in postmenopausal women. We analyzed humeri from monkeys that were either SHAM-operated or ovariectomized. Raman microspectroscopic analysis was performed as a function of tissue age based on the presence of multiple fluorescent double labels, to determine whether bone compositional properties (mineral/matrix ratio, tissue water, glycosaminoglycan, lipid, and pyridinoline contents, and mineral maturity/crystallinity) are similar between periosteal, osteonal, and endosteal surfaces, as well as to determine the effects of ovarian hormone depletion on them. The results indicate that mineral and organic matrix characteristics, and kinetics of mineral and organic matrix modifications as a function of tissue age are different at periosteal vs. osteonal and endosteal surfaces. Ovarian hormone depletion affects the three cortical surfaces (periosteal, osteonal, endosteal) differently. While ovarian hormone depletion does not significantly affect the quality of either the osteoid or the most recently mineralized tissue, it significantly affects the rate of subsequent mineral accumulation, as well as the kinetics of organic matrix modifications, culminating in significant differences within interstitial bone. These results highlight the complexity of the cortical bone compartments, add to existing knowledge on the effects of ovarian hormone depletion on local cortical bone properties, and may contribute to a better understanding of the location specific action of drugs used in the management of postmenopausal osteoporosis.

Effects of adaptation on numerosity decoding in the human brain.

Psychophysical studies have shown that numerosity is a sensory attribute susceptible to adaptation. Neuroimaging studies have reported that, at least for relatively low numbers, numerosity can be accurately discriminated in the intra-parietal sulcus. Here we developed a novel rapid adaptation paradigm where adapting and test stimuli are separated by pauses sufficient to dissociate their BOLD activity. We used multivariate pattern recognition to classify brain activity evoked by non-symbolic numbers over a wide range (20-80), both before and after psychophysical adaptation to the highest numerosity. Adaptation caused underestimation of all lower numerosities, and decreased slightly the average BOLD responses in V1 and IPS. Using support vector machine, we showed that the BOLD response of IPS, but not in V1, classified numerosity well, both when tested before and after adaptation. However, there was no transfer from training pre-adaptation responses to testing post-adaptation, and vice versa, indicating that adaptation changes the neuronal representation of the numerosity. Interestingly, decoding was more accurate after adaptation, and the amount of improvement correlated with the amount of perceptual underestimation of numerosity across subjects. These results suggest that numerosity adaptation acts directly on IPS, rather than indirectly via other low-level stimulus parameters analysis, and that adaptation improves the capacity to discriminate numerosity.

Adaptation to number operates on perceived rather than physical numerosity.

Humans share with many animals a number sense, the ability to estimate rapidly the approximate number of items in a scene. Recent work has shown that like many other perceptual attributes, numerosity is susceptible to adaptation. It is not clear, however, whether adaptation works directly on mechanisms selective to numerosity, or via related mechanisms, such as those tuned to texture density. To disentangle this issue we measured adaptation of numerosity of 10 pairs of connected dots, as connecting dots makes them appear to be less numerous than unconnected dots. Adaptation to a 20-dot pattern (same number of dots as the test) caused robust reduction in apparent numerosity of the connected-dot pattern, but not of the unconnected dot-pattern. This suggests that adaptation to numerosity, at least for relatively sparse dot-pattern, occurs at neural levels encoding perceived numerosity, rather than at lower levels responding to the number of elements in the scene.

A low-cost and versatile system for projecting wide-field visual stimuli within fMRI scanners.

We have constructed and tested a custom-made magnetic-imaging-compatible visual projection system designed to project on a very wide visual field (~80°). A standard projector was modified with a coupling lens, projecting images into the termination of an image fiber. The other termination of the fiber was placed in the 3-T scanner room with a projection lens, which projected the images relayed by the fiber onto a screen over the head coil, viewed by a participant wearing magnifying goggles. To validate the system, wide-field stimuli were presented in order to identify retinotopic visual areas. The results showed that this low-cost and versatile optical system may be a valuable tool to map visual areas in the brain that process peripheral receptive fields.

Proposed pathogenesis for atypical femoral fractures: lessons from materials research.

Atypical femoral fractures (AFFs) have been well defined clinically and epidemiologically. Less clear are the underlying mechanisms responsible. This commentary points out the likely sources of decreased resistance to fracture using lessons from bone material studies and biomechanics. We hypothesize that the key element in the cascade of events leading to failure of the largest and strongest bone in the human body is long-term suppression of normal bone turnover caused by exposure to potent anti-remodeling agents, most notably the bisphosphonates (BPs). Suppressed bone turnover produces changes in bone that alter its material quality and these changes could lead to adverse effects on its mechanical function. At the submicroscopic [<1 µm] level of collagen fibrils, suppression of bone turnover allows continued addition of non-enzymatic cross links that can reduce collagen's plasticity and this in turn contributes to reduced bone toughness. Further, adverse changes in hydroxyapatite crystalline structure and composition can occur, perhaps increasing collagen's brittleness. At the microscopic level [~1-500 µm] of the bone-matrix structure, suppressed bone turnover allows full mineralization of cortical bone osteons and results in a microstructure of bone that is more homogeneous. Both brittleness and loss of heterogeneity allow greater progression of microscopic cracks that can occur with usual physical activity; in crack mechanical terms, normal mechanisms that dissipate crack tip growth energy are greatly reduced and crack progression is less impeded. Further, the targeted repair of cracks by newly activated BMUs appears to be preferentially suppressed by BPs. We further hypothesize that it is not necessary to have accumulation of many cracks to produce an AFF, just one that progresses - one that is not stopped by bone's several protective mechanisms and is allowed to penetrate through a homogeneous environment. The remarkable straight transverse fracture line is an indicator of the slow progression of a "mother crack" and the failure of usual mechanisms to bridge or deflect the crack. Research in AFF mechanisms has been focused at the organ level, describing the clinical presentation and radiologic appearance. Although today we have not yet connected all the dots in the pathophysiology of BP-induced AFF, recent advances in measuring bone mechanical qualities at the submicroscopic and tissue levels allow us to explain how spontaneous catastrophic failure of the femur can occur.

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.

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.

Issues in modern bone histomorphometry.

This review reports on proceedings of a bone histomorphometry session conducted at the Fortieth International IBMS Sun Valley Skeletal Tissue Biology Workshop held on August 1, 2010. The session was prompted by recent technical problems encountered in conducting histomorphometry on bone biopsies from humans and animals treated with anti-remodeling agents such as bisphosphonates and RANKL antibodies. These agents reduce remodeling substantially, and thus cause problems in calculating bone remodeling dynamics using in vivo fluorochrome labeling. The tissue specimens often contain few or no fluorochrome labels, and thus create statistical and other problems in analyzing variables such as mineral apposition rates, mineralizing surface and bone formation rates. The conference attendees discussed these problems and their resolutions, and the proceedings reported here summarize their discussions and recommendations.

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.

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.

The influence of vitamin E on immune function and response to vaccination in older horses.

Horses have an increased susceptibility to infection because of a decline in immune function with advancing age. Vitamin E has been found to play a key role in normal immune system function. The purpose of the study was to examine the effect of vitamin E supplementation on immune function and response to vaccination in older horses. Predominantly older horses (18.9 +/- 1.3 yr, range 7 to 26 yr; 523 +/- 38 kg of BW) were supplemented orally once daily for 16 wk with either all-rac-alpha-tocopheryl acetate (15 IU/kg of BW; n = 8) or a placebo (n = 8). One horse from each group was removed from the study for reasons not related to the study. Serum alpha-tocopherol concentration, neutrophil and monocyte bacterial killing ability, lysozyme activity, immunoglobulin concentration (IgG(a), IgG(b), IgG(T), and IgM), and neutralizing antibody production to West Nile virus vaccination were determined. The overall serum alpha-tocopherol concentration of the vitamin E-supplemented horses was greater than that of placebo-supplemented horses (P < 0.001). Bacterial killing capacity of monocytes and neutrophils increased in the vitamin E-supplemented horses (P < 0.05). Vitamin E-supplemented horses had greater serum IgG(a) (P < 0.001) and IgG(T) (P = 0.003) concentrations but produced less serum IgG(b) (P = 0.023) than placebo-supplemented horses. There was no effect of vitamin E supplementation on IgM production. The neutralizing antibody response to vaccination against West Nile virus was unaffected by vitamin E supplementation. There was a continuous increase in serum lysozyme concentration in placebo-supplemented horses, whereas serum lysozyme concentration did not increase until wk 12 in vitamin E-supplemented horses. In conclusion, vitamin E supplementation of predominantly older horses differentially modulated general cell-mediated and humoral immune function. Further research is needed to fully understand the effect of vitamin E on the immune function of horses.

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.