Six months of voluntary alcohol consumption in male cynomolgus macaques reduces intracortical bone porosity without altering mineralization or mechanical properties.

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = -0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

Bone stress injuries.

Bone stress injuries, including stress fractures, are overuse injuries that lead to substantial morbidity in active individuals. These injuries occur when excessive repetitive loads are introduced to a generally normal skeleton. Although the precise mechanisms for bone stress injuries are not completely understood, the prevailing theory is that an imbalance in bone metabolism favours microdamage accumulation over its removal and replacement with new bone via targeted remodelling. Diagnosis is achieved by a combination of patient history and physical examination, with imaging used for confirmation. Management of bone stress injuries is guided by their location and consequent risk of healing complications. Bone stress injuries at low-risk sites typically heal with activity modification followed by progressive loading and return to activity. Additional treatment approaches include non-weight-bearing immobilization, medications or surgery, but these approaches are usually limited to managing bone stress injuries that occur at high-risk sites. A comprehensive strategy that integrates anatomical, biomechanical and biological risk factors has the potential to improve the understanding of these injuries and aid in their prevention and management.

AGN1 implant material to treat bone loss: Resorbable implant forms normal bone with and without alendronate in a canine critical size humeral defect model.

BACKGROUND: Fractures secondary to osteoporosis, particularly those of the hip and spine, are a major public health concern with high social and economic costs. The Local Osteo-Enhancement Procedure (LOEP) is an approach intended to strengthen skeletal areas that are at the highest risk for fracture due to osteoporosis. LOEP involves the implantation of AGN1, a triphasic, calcium-based, osteoconductive material which is then resorbed and replaced by bone. Since alendronate is the most prescribed osteoporotic treatment, the purpose of this canine study is to determine if the newly formed bone has the same properties as normal bone and whether alendronate treatment impacts AGN1 resorption and replacement with bone. METHODS: Sixty skeletally mature male hounds (24-38 kg) were evenly divided between alendronate (0.2 mg/kg/day) and non-alendronate treatment groups. A critical-size core bone defect created in one proximal humerus was implanted with AGN1 while the contralateral non-operated humerus served as a paired control in each animal. Animals were sacrificed 13, 26, and 52 weeks post-operatively (10 per treatment per timepoint). The control and treatment site bone specimens from each animal were examined using radiographic, histomorphometric, and biomechanical techniques. Results between alendronate-treated and non-alendronate-treated animals were compared as groups. RESULTS: AGN1 implant material was consistently resorbed and replaced by bone in all animals. At 52 weeks, only minimal residual implant material could be detected (0.9 ± 2.3% non-alendronate group; 2.2 ± 3.1% alendronate group), and new bone filled the defects in both the non-alendronate and alendronate groups. At 13 and 26 weeks, microCT revealed the newly formed bone in the defects had significantly higher trabecular bone volume and number connectivity than control bone in both groups. Mechanical testing demonstrated that the new bone had ultimate compressive strength and modulus equivalent to control bone as early as 13 weeks post-surgery which was maintained to 52 weeks in both groups. CONCLUSIONS: In this canine critical-sized humeral core defect model, AGN1 was progressively replaced by normal bone as evaluated by all outcome measures. Concurrent alendronate therapy did not significantly impact AGN1 resorption or new bone formation. These results demonstrate that AGN1 can be used in conjunction with alendronate in non-osteoporotic animals. CLINICAL RELEVANCE: This study suggests that the AGN1 implant material demonstrates potential for local restoration of bone in critical-size core defects, and that the material is compatible with alendronate drug therapy. Further studies will be required to determine if these results apply to other osteoporosis medications.

Micropetrosis in hemodialysis patients.

Micropetrosis develops as a result of stagnation of calcium, phosphorus and bone fluid, which appears as highly mineralized bone area in the osteocytic perilacunar/canalicular system regardless of bone turnover of the patients. And microcracks are predisposed to increase in these areas, which leads to increased bone fragility. However, micropetrosis of hemodialysis (HD) patients has not been discussed at all. Micropetrosis area per bone area (Mp.Ar/B·Ar) and osteocyte number per micropetrosis area (Ot.N/Mp.Ar) were measured in nine HD patients with renal hyperparathyroidism (Group I), twelve patients with hypoparathyroidism within 1 year after the treatment of renal hyperparathyroidism (Group II) and seven patients suffering from hypoparathyroidism for over two years (Group III). And bone mineral density (BMD) and tissue mineral density (TMD) were calculated using µCT to evaluate bone mineral content of iliac bone of the patients. These parameters were compared among the three groups. Only Mp.Ar/B·Ar was statistically greater in Group II and III compared to Group I in the parameters of bone mineral content and micropetrosis. However, the other parameters were not statistically different among the three groups. In long-term HD patients, BMD and TMD may be modified by the causes of renal insufficiency and the treatment of renal bone disease. We concluded that Mp.Ar/B·Ar was greater in patients with long-term hypoparathyroidism than both those with short-term hypoparathyroidism and with renal hyperparathyroidism. Special attention should be paid to avoid long-term hypoparathyroidism of the patients from the view point of increased fracture risk caused by increased micropetrosis area.

Effects of anti-resorptive treatment on the material properties of individual canine trabeculae in cyclic tensile tests.

Osteoporosis is defined as a decrease of bone mass and strength, as well as an increase in fracture risk. It is conventionally treated with antiresorptive drugs, such as bisphosphonates (BPs) and selective estrogen receptor modulators (SERMs). Although both drug types successfully decrease the risk of bone fractures, their effect on bone mass and strength is different. For instance, BP treatment causes an increase of bone mass, stiffness and strength of whole bones, whereas SERM treatment causes only small (4%) increases of bone mass, but increased bone toughness. Such improved mechanical behavior of whole bones can be potentially related to the bone mass, bone structure or material changes. While bone mass and architecture have already been investigated previously, little is known about the mechanical behavior at the tissue/material level, especially of trabecular bone. As such, the goal of the work presented here was to fill this gap by performing cyclic tensile tests in a wet, close to physiologic environment of individual trabeculae retrieved from the vertebrae of beagle dogs treated with alendronate (a BP), raloxifene (a SERM) or without treatments. Identification of material properties was performed with a previously developed rheological model and of mechanical properties via fitting of envelope curves. Additionally, tissue mineral density (TMD) and microdamage formation were analyzed. Alendronate treatment resulted in a higher trabecular tissue stiffness and strength, associated with higher levels of TMD. In contrast, raloxifene treatment caused a higher trabecular toughness, pre-dominantly in the post-yield region. Microdamage formation during testing was not affected by either anti-resorptive treatment regimens. These findings highlight that the improved mechanical behavior of whole bones after anti-resorptive treatment is at least partly caused by improved material properties, with different mechanisms for alendronate and raloxifene. This study further shows the power of performing a mechanical characterization of trabecular bone at the level of individual trabeculae for better understanding of clinically relevant mechanical behavior of bone.

Skeletal Protection and Promotion of Microbiome Diversity by Dietary Boosting of the Endogenous Antioxidant Response.

There is an unmet need for interventions with better compliance that prevent the adverse effects of sex steroid deficiency on the musculoskeletal system. We identified a blueberry cultivar (Montgomerym [Mont]) that added to the diet protects female mice from musculoskeletal loss and body weight changes induced by ovariectomy. Mont, but not other blueberries, increased the endogenous antioxidant response by bypassing the traditional antioxidant transcription factor Nrf2 and without activating estrogen receptor canonical signaling. Remarkably, Mont did not protect the male skeleton from androgen-induced bone loss. Moreover, Mont increased the variety of bacterial communities in the gut microbiome (α-diversity) more in female than in male mice; shifted the phylogenetic relatedness of bacterial communities (ß-diversity) further in females than males; and increased the prevalence of the taxon Ruminococcus1 in females but not males. Therefore, this nonpharmacologic intervention (i) protects from estrogen but not androgen deficiency; (ii) preserves bone, skeletal muscle, and body composition; (iii) elicits antioxidant defense responses independently of classical antioxidant/estrogenic signaling; and (iv) increases gut microbiome diversity toward a healthier signature. These findings highlight the impact of nutrition on musculoskeletal and gut microbiome homeostasis and support the precision medicine principle of tailoring dietary interventions to patient individualities, like sex. © 2020 American Society for Bone and Mineral Research (ASBMR).

Fifty years of bisphosphonates: What are their mechanical effects on bone?

After fifty years of experience with several generations of bisphosphonates (BPs), and 25 years after these drugs were approved for use in humans, their mechanical effects on bone are still not fully understood. Certainly, these drugs have transformed the treatment of osteoporosis in both men and women. There is no question that they do prevent fractures related to low bone mass, and there is widespread agreement that they increase strength and stiffness of the vertebrae. There is less consensus, however, about their effects on cortical bone, or on bone tissue properties in either trabecular or cortical bone, or their effects with longer periods of treatment. The consensus of most studies, both those based on ovariectomized and intact animal models and on testing of human bone, is that long-term treatment and/or high doses with certain BPs make the bone tissue more brittle and less tough. This translates into reduced energy to fracture and potentially a shorter bone fatigue life. Many studies have been done, but Interpretation of the results of these studies is complicated by variations in which BP is used, the animal model used, dose, duration, and methods of testing. Duration effects and effects on impact properties of bone are gaps that should be filled with additional testing.

The Importance of Biologically Active Vitamin D for Mineralization by Osteocytes After Parathyroidectomy for Renal Hyperparathyroidism.

Hypomineralized matrix is a factor determining bone mineral density. Increased perilacunar hypomineralized bone area is caused by reduced mineralization by osteocytes. The importance of vitamin D in the mineralization by osteocytes was investigated in hemodialysis patients who underwent total parathyroidectomy (PTX) with immediate autotransplantation of diffuse hyperplastic parathyroid tissue. No previous reports on this subject exist. The study was conducted in 19 patients with renal hyperparathyroidism treated with PTX. In 15 patients, the serum calcium levels were maintained by subsequent administration of alfacalcidol (2.0 µg/day), i.v. calcium gluconate, and oral calcium carbonate for 4 weeks after PTX (group I). This was followed in a subset of 4 patients in group I by a reduced dose of 0.5 µg/day until 1 year following PTX; this was defined as group II. In the remaining 4 patients, who were not in group I, the serum calcium (Ca) levels were maintained without subsequent administration of alfacalcidol (group III). Transiliac bone biopsy specimens were obtained in all groups before and 3 or 4 weeks after PTX to evaluate the change of the hypomineralized bone area. In addition, patients from group II underwent a third bone biopsy 1 year following PTX. A significant decrease of perilacunar hypomineralized bone area was observed 3 or 4 weeks after PTX in all group I and II patients. The area was increased again in the group II patients 1 year following PTX. In group III patients, an increase of the hypomineralized bone area was observed 4 weeks after PTX. The maintenance of a proper dose of vitamin D is necessary for mineralization by osteocytes, which is important to increase bone mineral density after PTX for renal hyperparathyroidism. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Voluntary Chronic Heavy Alcohol Consumption in Male Rhesus Macaques Suppresses Cancellous Bone Formation and Increases Bone Marrow Adiposity.

BACKGROUND: Chronic heavy alcohol consumption is an established risk factor for bone fracture, but comorbidities associated with alcohol intake may contribute to increased fracture rates in alcohol abusers. To address the specific effects of alcohol on bone, we used a nonhuman primate model and evaluated voluntary alcohol consumption on: (i) global markers of bone turnover in blood and (ii) cancellous bone mass, density, microarchitecture, turnover, and microdamage in lumbar vertebra. METHODS: Following a 4-month induction period, 6-year-old male rhesus macaques (Macaca mulatta, n = 13) voluntarily self-administered water or ethanol (EtOH; 4% w/v) for 22 h/d, 7 d/wk, for a total of 12 months. Control animals (n = 9) consumed an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3 days prior to sacrifice to label mineralizing bone surfaces. Global skeletal response to EtOH was evaluated by measuring plasma osteocalcin and carboxyterminal collagen cross-links (CTX). Local response was evaluated in lumbar vertebra using dual-energy X-ray absorptiometry, microcomputed tomography, static and dynamic histomorphometry, and histological assessment of microdamage. RESULTS: Monkeys in the EtOH group consumed an average of 2.8 ± 0.2 (mean ± SE) g/kg/d of EtOH (30 ± 2% of total calories), resulting in an average blood EtOH concentration of 88.3 ± 8.8 mg/dl 7 hours after the session onset. Plasma CTX and osteocalcin tended to be lower in EtOH-consuming monkeys compared to controls. Significant differences in bone mineral density in lumbar vertebrae 1 to 4 were not detected with treatment. However, cancellous bone volume fraction (in cores biopsied from the central region of the third vertebral body) was lower in EtOH-consuming monkeys compared to controls. Furthermore, EtOH-consuming monkeys had lower osteoblast perimeter and mineralizing perimeter, no significant difference in osteoclast perimeter, and higher bone marrow adiposity than controls. No significant differences between groups were detected in microcrack density (2nd lumbar vertebra). CONCLUSIONS: Voluntary chronic heavy EtOH consumption reduces cancellous bone formation in lumbar vertebra by decreasing osteoblast-lined bone perimeter, a response associated with an increase in bone marrow adiposity.

Stress concentrations and bone microdamage: John Currey's contributions to understanding the initiation and arrest of cracks in bone.

The microarchitecture of bone tissue presents many features that could act as stress concentrators for the initiation of bone microdamage. This was first identified by John Currey in a seminal paper in 1962 in which he presented the mechanical and biological evidence for stress concentrations at the bone surface, within the bone through the action of stiffness differentials between architectural features including between lamellae, and at the level of the lacunar and canalicular walls. Those early observations set the stage to consider how microscopic damage to bone tissue might affect the properties of bone at a time when most in the scientific community dismissed microcracks in bone as artifact. Evidence collected in the nearly 60 years since those important initial observations suggest that some of these architectural features in bone tissue are more effective as crack arrestors than as crack initiators. Sites of higher mineralization in the bone matrix, particularly interstitial sites in both cortical and trabecular bone, may serve preferentially as locations for crack initiation, whereas those boundaries identified by Currey as both stress concentrators and stress arrestors are more effective at stopping cracks than at initiating them.

Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality.

A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion: a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. The expression of matrix genes that contribute to bone material-level mechanical properties was elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.

Microcrack-associated bone remodeling is rarely observed in biopsies from athletes with medial tibial stress syndrome.

The pathology of medial tibial stress syndrome (MTSS) is unknown. Studies suggest that MTSS is a bony overload injury, but histological evidence is sparse. The presence of microdamage, and its potential association with targeted remodeling, could provide evidence for the pathogenesis of MTSS. Understanding the pathology underlying MTSS could contribute to effective preventative and therapeutic interventions for MTSS. Our aim was to retrospectively evaluate biopsies, previously taken from the painful area in athletes with MTSS, for the presence of linear microcracks, diffuse microdamage and remodeling. Biopsies, previously taken from athletes with MTSS, were evaluated at the Department of Anatomy and Cell Biology at the Indiana University. After preparing the specimens by en bloc staining, one investigator evaluated the presence of linear microcracks, diffuse microdamage and remodeling in the specimens. A total of six biopsies were evaluated for the presence of microdamage and remodeling. Linear microcracks were found in 4 out of 6 biopsies. Cracking in one of these specimens was artefactual due to the biopsy procedure. No diffuse microdamage was seen in any of the specimens, and only one potential remodeling front in association with the microcracks. We found only linear microcracks in vivo in biopsies taken from the painful area in 50% of the athletes with MTSS, consistent with the relationship between linear cracks and fatigue-associated overloading of bone. The nearly universal absence of a repair reaction was notable. This suggests that unrepaired microdamage accumulation may underlie the pathophysiological basis for MTSS in athletes.

Changes in bone matrix properties with aging.

It is well known that bone loss accompanies aging in both men and women and contributes to skeletal fragility in the older population, but changes that occur to the bone tissue matrix itself are less well known. These changes in bone quality aggravate the skeletal fragility associated with loss of bone mass. Bone tissue quality is affected by age-related changes in bone mineral, collagen and its cross-linking profiles, water compartments and even non-collagenous proteins. It is commonly assumed that greater tissue mineralization accompanies aging as bone turnover slows down in elderly individuals, but the data for this are weak. However, there may be changes in the quality of the mineral crystals, and the substitutions found within the crystal. Both enzymatically-mediated and non-enzymatically-mediated collagen cross-links multiply with age. The former tend to make the bone stiffer and stronger, but the latter, while making the bone stiffer can also make it more brittle and more likely to fracture. Bone pore water that is not bound to collagen or mineral increases with age as bone mass is lost, but water that is bound to collagen and mineral declines with age. These changes contribute to skeletal fragility by reducing the amount that bone can deform before fracturing. Finally, non-collagenous proteins have physical properties that can alter matrix mechanical properties and can also have molecular signaling functions that regulate bone remodeling. Whether these change with age, how they change, and how this affects skeletal fragility with aging is still largely a black box, and requires much more investigation. The roles of any of these factors in skeletal fragility are difficult to assess clinically as there is no easy or economical way to evaluate them, but a picture of fragility in the aging skeleton is incomplete without them.

Teriparatide Treatment Increases Mineral Content and Volume in Cortical and Trabecular Bone of Iliac Crest: A Comparison of Infrared Imaging With X-Ray-Based Bone Assessment Techniques.

Teriparatide increases bone mass primarily through remodeling of older or damaged bone and abundant replacement with new mineralizing bone. This post hoc analysis investigated whether dual-energy X-ray absorptiometric (DXA) areal bone mineral density (aBMD) measurement adequately reflects changes of mineral and organic matrix content in cortical and trabecular bone. Paired biopsies and aBMD measurements were obtained before and at end of 2 years of teriparatide treatment from postmenopausal women with osteoporosis who were either alendronate pretreated (mean, 57.5 months) or osteoporosis-treatment naive. Biopsies were assessed by micro-computed tomography (µCT) to calculate mean cortical width (Ct.Wi), cortical area (Ct.Ar), and trabecular bone volume fraction (BV/TV). Fourier transformed infrared imaging (pixel size ∼6.3 × 6.3 µm2 ) was utilized to calculate mineral and organic matrix density (mean absorption/pixel), as well as total mineral and organic contents of cortical and cancellous compartments (sum of all pixels in the compartment). Effect of pretreatment over time was analyzed using mixed model repeated measures. µCT derived Ct.Wi and BV/TV increased, accompanied by similar increases in the overall mineral contents of their respective bone compartments. Mineral density did not change. Marked increases in the total content of both mineral and organic matrix associated with volumetric growth in both compartments consistently exceeded those of aBMD. Increases in organic matrix exceeded increases in mineral content in both cortical and trabecular compartments. For percent changes, only change in Ct.Wi correlated to change in femoral neck aBMD (r = .38, p = 0.043), whereas no other significant correlations of Ct.Wi or BV/TV with lumbar spine, total hip, or femoral neck aBMD were demonstrable. These data indicate that 2 years of teriparatide treatment leads to an increased bone organic matrix and mineral content in the iliac crest. The magnitude of these increases in the iliac crest were not detected with conventional aBMD measurements at other skeletal sites. © 2018 American Society for Bone and Mineral Research.

Osteocytic perilacunar/canalicular turnover in hemodialysis patients with high and low serum PTH levels.

Osteocytic perilacunar/canalicular turnover in hemodialysis patients has not yet been reported. Osteocyte lacunae in lamellar bone and woven bone were classified as eroded surface-, osteoid surface-, and quiescent surface-predominant osteocyte lacunae (ES-Lc, OS-Lc, QS-Lc, respectively) in 55 hemodialysis patients with either high- (n = 45) or low- (n = 10) parathyroid hormone levels, and 19 control subjects without chronic kidney disease. We calculated the area and number of ES-Lc, OS-Lc, and QS-Lc. The mineralized surface on the osteocyte lacunar walls was measured in each group, and compared among the three groups. The shapes of the osteocyte lacunar walls were validated by backscattered electron microscopy. While the number of ES-Lc per bone area (N.ES-Lc/B.Ar) was higher than the number of OS-Lc per bone area (N.OS-Lc/B.Ar) in all groups, N.ES-Lc/B.Ar and N.OS-Lc/B.Ar were greater in high-parathyroid hormone group than in low-parathyroid hormone and control groups. The total volume of ES-Lc per bone area (ES-Lc.Ar/B.Ar) was greater than the total volume of OS-Lc per bone area (OS-Lc.Ar/B.Ar) in both parathyroid hormone groups. However, both lacunar erosion and lacunar formation increased proportionally, suggesting that global coupling between them was maintained. N.ES-Lc/B.Ar was higher in woven bone than in lamellar bone. The rate of OS-Lc stained by tetracycline hydrochloride, the mineralized lacunar surface and the mean area of OS-Lc with Tc obtained from both parathyroid hormone groups were greater than those in the control group. We conclude that osteocytic perilacunar/canalicular turnover is increased in hemodialysis patients with high parathyroid hormone levels. Osteocytic perilacunar/canalicular turnover depends, at least in part, on serum parathyroid hormone level. However, the ideal PTH level for osteocytic perilacunar/canalicular turnover could not be determined but osteocytic osteolysis was predominant in both the high- and low-PTH groups in this study. Thus, attention should be paid to bone loss from the viewpoint of osteocytic perilacunar/canalicular turnover in hemodialysis patients.

Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification.

Approximately 10% of all bone fractures do not heal, resulting in patient morbidity and healthcare costs. However, no pharmacological treatments are currently available to promote efficient bone healing. Inhibition of Ca2+ /calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) reverses age-associated loss of trabecular and cortical bone volume and strength in mice. In the current study, we investigated the role of CaMKK2 in bone fracture healing and show that its pharmacological inhibition using STO-609 accelerates early cellular and molecular events associated with endochondral ossification, resulting in a more rapid and efficient healing of the fracture. Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus. Early deposition of primary bone by osteoblasts resulted in STO-609-treated mice possessing significantly higher callus bone volume by 14 days following fracture. Subsequent rapid maturation of the bone matrix bestowed fractured bones in STO-609-treated animals with significantly higher torsional strength and stiffness by 28 days postinjury, indicating accelerated healing of the fracture. Previous studies indicate that fixed and closed femoral fractures in the mice take 35 days to fully heal without treatment. Therefore, our data suggest that STO-609 potentiates a 20% acceleration of the bone healing process. Moreover, inhibiting CaMKK2 also imparted higher mechanical strength and stiffness at the contralateral cortical bone within 4 weeks of treatment. Taken together, the data presented here underscore the therapeutic potential of targeting CaMKK2 to promote efficacious and rapid healing of bone fractures and as a mechanism to strengthen normal bones. © 2018 American Society for Bone and Mineral Research.

Effects of combination treatment with alendronate and raloxifene on skeletal properties in a beagle dog model.

A growing number of studies have investigated combination treatment as an approach to treat bone disease. The goal of this study was to investigate the combination of alendronate and raloxifene with a particular focus on mechanical properties. To achieve this goal we utilized a large animal model, the beagle dog, used previously by our laboratory to study both alendronate and raloxifene monotherapies. Forty-eight skeletally mature female beagles (1-2 years old) received daily oral treatment: saline vehicle (VEH), alendronate (ALN), raloxifene (RAL) or both ALN and RAL. After 6 and 12 months of treatment, all animals underwent assessment of bone material properties using in vivo reference point indentation (RPI) and skeletal hydration using ultra-short echo magnetic resonance imaging (UTE-MRI). End point measures include imaging, histomorphometry, and mechanical properties. Bone formation rate was significantly lower in iliac crest trabecular bone of animals treated with ALN (-71%) and ALN+RAL (-81%) compared to VEH. In vivo assessment of properties by RPI yielded minimal differences between groups while UTE-MRI showed a RAL and RAL+ALN treatment regimens resulted in significantly higher bound water compared to VEH (+23 and +18%, respectively). There was no significant difference among groups for DXA- or CT-based measures lumbar vertebra, or femoral diaphysis. Ribs of RAL-treated animals were smaller and less dense compared to VEH and although mechanical properties were lower the material-level properties were equivalent to normal. In conclusion, we present a suite of data in a beagle dog model treated for one year with clinically-relevant doses of alendronate and raloxifene monotherapies or combination treatment with both agents. Despite the expected effects on bone remodeling, our study did not find the expected benefit of ALN to BMD or structural mechanical properties, and thus the viability of the combination therapy remains unclear.

Improving Combination Osteoporosis Therapy in a Preclinical Model of Heightened Osteoanabolism.

Combining anticatabolic agents with parathyroid hormone (PTH) to enhance bone mass has yielded mixed results in osteoporosis patients. Toward the goal of enhancing the efficacy of these regimens, we tested their utility in combination with loss of the transcription factor Nmp4 because disabling this gene amplifies PTH-induced increases in trabecular bone in mice by boosting osteoblast secretory activity. We addressed whether combining a sustained anabolic response with an anticatabolic results in superior bone acquisition compared with PTH monotherapy. Additionally, we inquired whether Nmp4 interferes with anticatabolic efficacy. Wild-type and Nmp4-/- mice were ovariectomized at 12 weeks of age, followed by therapy regimens, administered from 16 to 24 weeks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Anabolic therapeutic efficacy generally corresponded with PTH + RAL = PTH + ZOL > PTH + ALN = PTH > vehicle control. Loss of Nmp4 enhanced femoral trabecular bone increases under PTH + RAL and PTH + ZOL. RAL and ZOL promoted bone restoration, but unexpectedly, loss of Nmp4 boosted RAL-induced increases in femoral trabecular bone. The combination of PTH, RAL, and loss of Nmp4 significantly increased bone marrow osteoprogenitor number, but did not affect adipogenesis or osteoclastogenesis. RAL, but not ZOL, increased osteoprogenitors in both genotypes. Nmp4 status did not influence bone serum marker responses to treatments, but Nmp4-/- mice as a group showed elevated levels of the bone formation marker osteocalcin. We conclude that the heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, in part by increasing hyperanabolic osteoprogenitors. Nmp4 provides a promising target pathway for identifying barriers to pharmacologically induced bone formation.