Anti-DKK1 antibody promotes bone fracture healing through activation of β-catenin signaling

In this study we investigated if Wnt/β-catenin signaling in mesenchymal progenitor cells plays a role in bone fracture repair and if DKK1-Ab promotes fracture healing through activation of β-catenin signaling. Unilateral open transverse tibial fractures were created in CD1 mice and in β-catenin^Prx1ER conditional knockout (KO) and Cre-negative control mice (C57BL/6 background). Bone fracture callus tissues were collected and analyzed by radiography, micro-CT (μCT), histology, biomechanical testing and gene expression analysis. The results demonstrated that treatment with DKK1-Ab promoted bone callus formation and increased mechanical strength during the fracture healing process in CD1 mice. DKK1-Ab enhanced fracture repair by activation of endochondral ossification. The normal rate of bone repair was delayed when the β-catenin gene was conditionally deleted in mesenchymal progenitor cells during the early stages of fracture healing. DKK1-Ab appeared to act through β-catenin signaling to enhance bone repair since the beneficial effect of DKK1-Ab was abrogated in β-catenin^Prx1ER conditional KO mice. Further understanding of the signaling mechanism of DKK1-Ab in bone formation and bone regeneration may facilitate the clinical translation of this anabolic agent into therapeutic intervention.     

Loss of Gi G‐Protein‐Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing

G‐protein‐coupled receptors (GPCRs) are key regulators of skeletal homeostasis and are likely important in fracture healing. Because GPCRs can activate multiple signaling pathways simultaneously, we used targeted disruption of G_i‐GPCR or activation of G_s‐GPCR pathways to test how each pathway functions in the skeleton. We previously demonstrated that blockade of G_i signaling by pertussis toxin (PTX) transgene expression in maturing osteoblastic cells enhanced cortical and trabecular bone formation and prevented age‐related bone loss in female mice. In addition, activation of G_s signaling by expressing the G_s‐coupled engineered receptor Rs1 in maturing osteoblastic cells induced massive trabecular bone formation but cortical bone loss. Here, we test our hypothesis that the G_i and G_s pathways also have distinct functions in fracture repair. We applied closed, nonstabilized tibial fractures to mice in which endogenous G_i signaling was inhibited by PTX, or to mice with activated G_s signaling mediated by Rs1. Blockade of endogenous G_i resulted in a smaller callus but increased bone formation in both young and old mice. PTX treatment decreased expression of Dkk1 and increased Lef1 mRNAs during fracture healing, suggesting a role for endogenous G_i signaling in maintaining Dkk1 expression and suppressing Wnt signaling. In contrast, adult mice with activated G_s signaling showed a slight increase in the initial callus size with increased callus bone formation. These results show that G_i blockade and G_s activation of the same osteoblastic lineage cell can induce different biological responses during fracture healing. Our findings also show that manipulating the GPCR/cAMP signaling pathway by selective timing of G_s and G_i‐GPCR activation may be important for optimizing fracture repair. © 2015 American Society for Bone and Mineral Research.     

Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity

Bone fractures remain a serious health burden and prevention and enhanced healing of fractures have been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1^+/−) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2^c/c;Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1^+/− mice were crossed with Bmp2^c/c;Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy.     

Gain-of-Function Lrp5 Mutation Improves Bone Mass and Strength and Delays Hyperglycemia in a Mouse Model of Insulin-Deficient Diabetes

High fracture rate and high circulating levels of the Wnt inhibitor, sclerostin, have been reported in diabetic patients. We studied the effects of Wnt signaling activation on bone health in a mouse model of insulin-deficient diabetes. We introduced the sclerostin-resistant Lrp5^A214V mutation, associated with high bone mass, in mice carrying the Ins2^Akita mutation (Akita), which results in loss of beta cells, insulin deficiency, and diabetes in males. Akita mice accrue less trabecular bone mass with age relative to wild type (WT). Double heterozygous Lrp5^A214V/Akita mutants have high trabecular bone mass and cortical thickness relative to WT animals, as do Lrp5^A214V single mutants. Likewise, the Lrp5^A214V mutation prevents deterioration of biomechanical properties occurring in Akita mice. Notably, Lrp5^A214V/Akita mice develop fasting hyperglycemia and glucose intolerance with a delay relative to Akita mice (7 to 8 vs. 5 to 6 weeks, respectively), despite lack of insulin production in both groups by 6 weeks of age. Although insulin sensitivity is partially preserved in double heterozygous Lrp5^A214V/Akita relative to Akita mutants up to 30 weeks of age, insulin-dependent phosphorylated protein kinase B (pAKT) activation in vitro is not altered by the Lrp5^A214V mutation. Although white adipose tissue depots are equally reduced in both compound and Akita mice, the Lrp5^A214V mutation prevents brown adipose tissue whitening that occurs in Akita mice. Thus, hyperactivation of Lrp5-dependent signaling fully protects bone mass and strength in prolonged hyperglycemia and improves peripheral glucose metabolism in an insulin independent manner. Wnt signaling activation represents an ideal therapeutic approach for diabetic patients at high risk of fracture. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

High Bone Mass–Causing Mutant LRP5 Receptors Are Resistant to Endogenous Inhibitors In Vivo

Certain missense mutations affecting LRP5 cause high bone mass (HBM) in humans. Based on in vitro evidence, HBM LRP5 receptors are thought to exert their effects by providing resistance to binding/inhibition of secreted LRP5 inhibitors such as sclerostin (SOST) and Dickkopf homolog‐1 (DKK1). We previously reported the creation of two Lrp5 HBM knock‐in mouse models, in which the human p.A214V or p.G171V missense mutations were knocked into the endogenous Lrp5 locus. To determine whether HBM knock‐in mice are resistant to SOST‐ or DKK1‐induced osteopenia, we bred Lrp5 HBM mice with transgenic mice that overexpress human SOST in osteocytes (^8kbDmp1‐SOST) or mouse DKK1 in osteoblasts and osteocytes (^2.3kbCol1a1‐Dkk1). We observed that the ^8kbDmp1‐SOST transgene significantly lowered whole‐body bone mineral density (BMD), bone mineral content (BMC), femoral and vertebral trabecular bone volume fraction (BV/TV), and periosteal bone‐formation rate (BFR) in wild‐type mice but not in mice with Lrp5 p.G171V and p.A214V alleles. The ^2.3kbCol1a1‐Dkk1 transgene significantly lowered whole‐body BMD, BMC, and vertebral BV/TV in wild‐type mice and affected p.A214V mice more than p.G171V mice. These in vivo data support in vitro studies regarding the mechanism of HBM‐causing mutations, and imply that HBM LRP5 receptors differ in their relative sensitivity to inhibition by SOST and DKK1. © 2015 American Society for Bone and Mineral Research.     

Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6.

Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the related gene Lrp6. We found that compound mutants had dose-dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development. INTRODUCTION: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co-receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed. MATERIALS AND METHODS: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and muCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture. RESULTS: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6(+/-);Lrp5(+/-) or Lrp6(+/-);Lrp5(-/-)) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6(+/-);Lrp5(-/-) mice were more severely affected than Lrp6(+/+);Lrp5(-/-) mice, whereas Lrp6(+/-);Lrp5(+/-) mice had statistically higher BMD than Lrp6(+/+);Lrp5(-/-) mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone. CONCLUSIONS: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.     

Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures.

Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the time-course of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling. INTRODUCTION: Since FDA approval of PTH [PTH(1-34); Forteo] as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. MATERIALS AND METHODS: Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 microg/kg PTH(1-34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. RESULTS: Quantitative muCT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized beta-catenin protein, a central feature of canonical Wnt signaling. CONCLUSIONS: These results showed that the PTH-mediated enhancement of fracture repair is primarily associated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways.     

Inhibition of beta‐catenin signaling by Pb leads to incomplete fracture healing

There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb‐exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb‐treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased β‐catenin levels. Reduction of β‐catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb‐exposed mice throughout fracture healing. β‐catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in β‐catenin signaling, suggesting the Wnt/β‐catenin as a pivotal pathway in the influence of Pb on fracture repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1397–1405, 2014.     

PTH stimulates bone formation in mice deficient in Lrp5.

Lrp5 deficiency decreases bone formation and results in low bone mass. This study evaluated the bone anabolic response to intermittent PTH treatment in Lrp5-deficient mice. Our results indicate that Lrp5 is not essential for the stimulatory effect of PTH on cancellous and cortical bone formation. INTRODUCTION: Low-density lipoprotein receptor-related protein 5 (Lrp5), a co-receptor in canonical Wnt signaling, increases osteoblast proliferation, differentiation, and function. The purpose of this study was to use Lrp5-deficient mice to evaluate the potential role of this gene in mediating the bone anabolic effects of PTH. MATERIALS AND METHODS: Adult wildtype (WT, 23 male and 25 female) and Lrp5 knockout (KO, 27 male and 26 female) mice were treated subcutaneously with either vehicle or 80 microg/kg human PTH(1-34) on alternate days for 6 weeks. Femoral BMC and BMD were determined using DXA. Lumbar vertebrae were processed for quantitative bone histomorphometry. Bone architecture was evaluated by microCT. Data were analyzed using a multiway ANOVA. RESULTS: Cancellous and cortical bone mass were decreased with Lrp5 deficiency. Compared with WT mice, cancellous bone volume in the distal femur and the lumbar vertebra in Lrp5 KO mice was 54% and 38% lower, respectively (p<0.0001), whereas femoral cortical thickness was 11% lower in the KO mice (p<0.0001). The decrease in cancellous bone volume in the lumbar vertebrae was associated with a 45% decrease in osteoblast surface (p<0.0001) and a comparable decrease in bone formation rate (p<0.0001). Osteoclast surface, an index of bone resorption, was 24% lower in Lrp5 KO compared with WT mice (p<0.007). Treatment of mice with PTH for 6 weeks resulted in a 59% increase in osteoblast surface (p<0.0001) and a 19% increase in osteoclast surface (p=0.053) in both genotypes, but did not augment cancellous bone volume in either genotype. Femur cortical thickness was 11% higher in PTH-treated mice in comparison with vehicle-treated mice (p<0.0001), regardless of genotype. CONCLUSIONS: Whereas disruption of Lrp5 results in decreased bone mass because of decreased bone formation, Lrp5 does not seem to be essential for the stimulatory effects of PTH on cancellous and cortical bone formation.     

Where Wnts Went: The Exploding Field of Lrp5 and Lrp6 Signaling in Bone

Wnt signaling has emerged as a central regulator of skeletal modeling and remodeling. Loss‐ or gain‐of‐function mutations in two Wnt co‐receptors, Lrp5 and (more recently) Lrp6, have drawn attention to the importance of the Wnt pathway in bone biology. This review summarizes our current understanding of how the Wnt pathway operates on bone and the implications this has for skeletal physiology and drug discovery. Over the past 9 yr, rapid advances have been made in our understanding of the cellular targets for Wnt signaling and of the important regulatory molecules in this metabolic pathway. Both canonical and noncanonical signaling pathways seem to be important for mediating the effects of Wnt in bone. A rapidly expanding catalog of genetically engineered mice has been used to establish the importance of downstream effector molecules (such as β‐catenin) in the Wnt pathway, as well as the critical role of endogenous inhibitors of Wnt signaling (such as Dkk1 and sclerostin) in bone metabolism. Indeed, regulation of sclerostin in osteocytes is emerging as an important final pathway for regulating bone anabolism in response to diverse trophic stimuli, from mechnotransduction to the anabolic actions of PTH. From the outset, it had been assumed that the effects of Wnt signaling in bone were caused by direct actions in osteoblast precursors, osteoblasts, and osteocytes. However, startling recent findings have challenged this view and suggest that a key target, at least in mice, is the duodenal enterochromaffin cell. There, Wnt signaling transduced by Lrp5 regulates serotonin synthesis, which acts in an endocrine fashion to regulate bone cell metabolism. It will take time to reconcile this new information with the considerable body of information we already have regarding the actions of Wnt in bone. The Wnt pathway has rapidly emerged as a therapeutic target for drug discovery. Neutralizing antibodies and small‐molecule inhibitors of endogenous Wnt inhibitors have shown early promise as bone anabolic agents. However, given the central role of the Wnt pathway in regulating growth and development in extraskeletal tissues, as well as our still rudimentary understanding of how this signaling cascade actually affects bone metabolism, considerable work will be needed to ensure the safety of these new therapies.     

The Anti‐Osteoanabolic Function of Sclerostin Is Blunted in Mice Carrying a High Bone Mass Mutation of Lrp5

Activating mutations of the putative Wnt co‐receptor Lrp5 or inactivating mutations of the secreted molecule Sclerostin cause excessive bone formation in mice and humans. Previous studies have suggested that Sclerostin functions as an Lrp5 antagonist, yet clear in vivo evidence was still missing, and alternative mechanisms have been discussed. Moreover, because osteoblast‐specific inactivation of β‐catenin, the major intracellular mediator of canonical Wnt signaling, primarily affected bone resorption, it remained questionable, whether Sclerostin truly acts as a Wnt signaling antagonist by interacting with Lrp5. In an attempt to address this relevant question, we generated a mouse model (Col1a1‐Sost) with transgenic overexpression of Sclerostin under the control of a 2.3‐kb Col1a1 promoter fragment. These mice displayed the expected low bone mass phenotype as a consequence of reduced bone formation. The Col1a1‐Sost mice were then crossed with two mouse lines carrying different high bone mass mutations of Lrp5 (Lrp5^A170V and Lrp5^G213V), both of them potentially interfering with Sclerostin binding. Using µCT‐scanning and histomorphometry we found that the anti‐osteoanabolic influence of Sclerostin overexpression was not observed in Lrp5^A213V/A213V mice and strongly reduced in Lrp5^A170V/A170V mice. As a control we applied the same strategy with mice overexpressing the transmembrane Wnt signaling antagonist Krm2 and found that the anti‐osteoanabolic influence of the Col1a1‐Krm2 transgene was not affected by either of the Lrp5 mutations. Taken together, our data support the concept that Sclerostin inhibits bone formation through Lrp5 interaction, yet their physiological relevance remains to be established. © 2015 American Society for Bone and Mineral Research.     

Genetic Variations in Bone Density, Histomorphometry, and Strength in Mice

The purpose of this study was to assess breed-related differences in bone histomorphometry, bone biomechanics, and serum biochemistry in three mouse breeds shown to differ in bone mineral density (BMD) (as measured by DXA) and bone mineral content (BMC). Femurs, tibiae, and sera were collected from 16-week-old C3H/HeJ {C3H}, C57BL/6J {BL6}, and DBA/2J {DBA}mice (n = 12/breed). Data collected included BMC and BMD (femora), histomorphometry of cancellous (distal femur) and cortical bone (diaphyseal tibiae and femora), bone strength (femora), and serum alkaline phosphatase (ALP). Consistent with previous reports, BMC and BMD were higher in C3H than in BL6 or DBA mice. The higher BMD in the C3H breed was associated with greater cancellous bone volume, cortical bone area, periosteal bone formation rate, biomechanical strength, and serum ALP. However, mid-diaphyseal total femoral and tibial cross-sectional area and moment of inertia were greatest in BL6, intermediate in C3H, and lowest in DBA mice. The specific distribution of cortical bone in C3H, BL6, DBA mice represents a difference in adaptive response to similar mechanical loads in these breeds. This difference in adaptive response may be intrinsic to the adaptive mechanism, or may be intrinsic to the bone tissue material properties. In either case, the bone-adaptive response to ordinary mechanical loads in the BL6 mice yields bones of lower mechanical efficiency (less stiffness per unit mass of bone tissue) and does not adapt as well as that of the C3H mice where the final product is a bone with greater resistance to bending under load. We suggest that the size, shape, and BMD of the bone are a result of breed-specific genetically regulated cellular mechanisms. Compared with the C3H mice, the lower BMD in BL6 mice is associated with long bones that are weaker because the larger cross-sectional area fails to compensate completely for their lower BMD and BMC. Received: 16 November 1999 / Accepted: 19 April 2000 / Online publication: 27 July 2000     

Bone regeneration is regulated by wnt signaling.

Tissue regeneration is increasingly viewed as reactivation of a developmental process that, when misappropriated, can lead to malignant growth. Therefore, understanding the molecular and cellular pathways that govern tissue regeneration provides a glimpse into normal development as well as insights into pathological conditions such as cancer. Herein, we studied the role of Wnt signaling in skeletal tissue regeneration. INTRODUCTION: Some adult tissues have the ability to regenerate, and among these, bone is one of the most remarkable. Bone exhibits a persistent, lifelong capacity to reform after injury, and continual bone regeneration is a prerequisite to maintaining bone mass and density. Even slight perturbations in bone regeneration can have profound consequences, as exemplified by conditions such as osteoporosis and delayed skeletal repair. Here, our goal was to determine the role of Wnts in adult bone regeneration. MATERIALS AND METHODS: Using TOPgal reporter mice, we found that damage to the skeleton instigated Wnt reporter activity, specifically at the site of injury. We used a skeletal injury model to show that Wnt inhibition, achieved through adenoviral expression of Dkk1 in the adult skeleton, prevented the differentiation of osteoprogenitor cells. RESULTS: As a result, injury-induced bone regeneration was reduced by 84% compared with controls. Constitutive activation of the Wnt pathway resulting from a mutation in the Lrp5 Wnt co-receptor results in high bone mass, but our experiments showed that this same point mutation caused a delay in bone regeneration. In these transgenic mice, osteoprogenitor cells in the injury site were maintained in a proliferative state and differentiation into osteoblasts was delayed. CONCLUSIONS: When considered together, these data provide a framework for understanding the roles of Wnt signaling in adult bone regeneration and suggest a feasible approach to treating clinical conditions where enhanced bone formation is desired.     

Comparison of Fracture Healing Among Different Inbred Mouse Strains

Quantitative trait locus analysis can be used to identify genes critically involved in biological processes. No such analysis has been applied to identifying genes that control bone fracture healing. To determine the feasibility of such an approach, healing of femur fractures was measured between C57BL/6, DBA/2, and C3H inbred strains of mice. Healing was assessed by radiography and histology and measured by histomorphometry and biomechanical testing. In all strains, radiographic bridging of the fracture was apparent after 3 weeks of healing. Histology showed that healing occurred through endochondral ossification in all strains. Histomorphometric measurements found more bone in the C57BL/6 fracture calluses 7 and 10 days after fracture. In contrast, more cartilage was present after 7 days in the C3H callus, which rapidly declined to levels less than those of C57BL/6 or DBA/2 mice by 14 days after fracture. An endochondral ossification index was calculated by multiplying the callus percent cartilage and bone areas as a measure of endochondral ossification. At 7 and 10 days after fracture, this value was higher in C57BL/6 mice. Using torsional mechanical testing, normalized structural and material properties of the C57BL/6 healing femurs were higher than values from the DBA/2 or C3H mice 4 weeks after fracture. The data indicate that fracture healing proceeds more rapidly in C57BL/6 mice and demonstrate that genetic variability significantly contributes to the process of bone regeneration. Large enough differences exist between C57BL/6 and DBA/2 or C3H mice to permit a quantitative trait locus analysis to identify genes controlling bone regeneration.     

Dickkopf‐1 regulates bone formation in young growing rodents and upon traumatic injury

The physiological role of Dickkopf‐1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1‐Ab) that blocked Dkk1 binding to both low density lipoprotein receptor‐related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1‐Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury. © 2011 American Society for Bone and Mineral Research     

Beta-catenin-dependent Wnt signaling in mandibular bone regeneration

Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.     

Relationship among bone mineral density, collagen composition, and biomechanical properties of callus in the healing of osteoporotic fracture

Objective： To study the change and relationship among bone mineral density （BMD）, collagen composition and biomechanical properties of the callus in the healing process of osteoporotic fracture. Methods： The osteoporotic rat model and fracture model were established through bilateral ovariectomy （OV＇X） and osteotomy of the middle shaft of the right hind tibiae, respectively. Ninety female SD rats were randomly divided into OVX group and sham group. With the samples of blood and callus, roentgenoraphic and histological observation were performed for the assessment of the healing progress of the fracture, and the serum concentration of TRAP-5b, proportion of type I collagen, BMD and biomechanical properties of the callus were measured. Results： The OVX group experienced a significant delay of fracture healing. The mean serum concentration of TRAP-5b of rats in the OVX group was much higher than that in the sham group after the operation （P 〈 0.05）, but the difference at the same time point after fracture was smaller than that before fracture （P 〈 0.05）. The BMD of the callus in both groups reached the peak value at the 6 th week after fracture while the proportion of the type I collagen and the biomechanical strength reached the peak at the 8th week. Conclusions. The deficiency of estrogen after the ovariectomy could induce the up-regulation of the osteoclasts activities, whereas the potency of further activation after fracture was depressed. Although the synthesis of collagen together with its mineralization determines the biomechanical properties of new bone, the accumulation of collagen could be assessed as an index in the prediction of biomechanical strength of bones independent of the bone mineral deposition.     

Oim mice exhibit altered femur and incisor mineral composition and decreased bone mineral density

To investigate the role of the pro alpha 2(I) collagen chains of type I collagen in mineralization we used the oim (osteogenesis imperfecta model) mouse as our model system. The oim/oim mouse (homozygous for a null mutation in its COL1A2 gene of type I collagen) fails to synthesize functional pro alpha 2(I) collagen chains, synthesizing only homotrimers of pro alpha 1(I) collagen chains. To evaluate the role of pro alpha 2(I) collagen in type I collagen structure/function in mineralized tissues, we examined age-matched oim/oim, heterozygous (oim/+), and wild-type (+/+) mouse femurs and incisors for mineral composition (calcium, phosphorus, magnesium, fluoride, sodium, potassium, and chloride) by neutron activation analyses (NAA), and bone mineral content (BMC) and bone mineral density (BMD) by dual-energy X-ray absorptiometry (DEXA) in a longitudinal study (7 weeks to 16 months of age). NAA demonstrated that oim/oim femurs had significant differences in magnesium, fluoride, and sodium content as compared with +/+ mouse femurs, and oim/oim teeth had significant differences in magnesium content as compared to +/+ teeth. The ratio of calcium to phosphate was also significantly reduced in the oim/oim mouse femurs (1.58 +/- 0.01) compared with +/+ femurs (1.63 +/- 0.01). DEXA demonstrated that oim/oim mice had significantly reduced BMC and BMD as compared to oim/+ and +/+ mice. Serum and urine calcium, magnesium, and phosphorus levels, and Ca(47) absorption across the gut were equivalent in oim/oim and +/+ mice, with no evidence of hypercalciuria. These studies suggest that the known decreased biomechanical properties of oim/oim bone reflect both altered mineral composition as well as the decreased BMD, which further suggests that the presence of alpha2(I) chains plays an important role in mineralization.