Zoledronate reverses mandibular bone loss in osteoprotegerin-deficient mice

Summary  To characterize the changes in osteoprotegerin-deficient (OPG−/−) mice mandibles and the possible mandibular bone loss prevention by zoledronate. This preventive effect in the mandible differed from that in the proximal tibia and was independent of the OPG pathway. Introduction  The study aimed to characterize both the changes in the mandible in osteoprotegerin-deficient (OPG^−/−) mice and possible mandibular bone loss prevention by zoledronate. Methods  Twenty-eight 6-week-old female mice (C57BL/6J), including OPG^−/− (n = 21) and wild-type (WT) (n = 7) mice, were assigned to four groups after 2 weeks of acclimatization to local vivarium conditions: wild mice with vehicle (WT group); OPG^−/− mice with vehicle (OPG^−/− group); and OPG^−/− mice that were subcutaneously injected with either 50 or 150 μg/kg zoledronate (Zol-50 and Zol-150 groups, respectively). Mice were sacrificed at 4 weeks after these treatments and after fasting for 12 h. Sera were harvested for biochemical analyses. The right mandible and tibia of each mouse were selected for microCT analysis. Student’s t-test was performed for comparisons of bone parameters at different sites in the WT group. Analysis of variance (ANOVA) was used to compare the biomarkers and bone parameters in the different treatment groups. Results  Serum bone-specific alkaline phosphatase (B-ALP) and tartrate-resistant acid phosphatase 5b (TRACP-5b) were significantly decreased in WT mice as compared to the levels in the OPG^−/− mice (P < 0.05). Zoledronate treatment decreased the high serum B-ALP activity observed in OPG^−/− mice to the levels seen in WT mice, while serum TRACP-5b concentrations were decreased to levels even lower than those in WT mice. There were substantial variations in BMD and microstructure of the mandibular and proximal tibial trabeculae. Mandibular bone loss was less affected by OPG gene deprivation than the proximal tibia was. Both zoledronate groups showed greater BMD, trabecular BV/TV, Tb.Th, Tb.N, and Conn.D and a significant decrease in Tb.Sp and SMI as compared to the findings in OPG^−/− mice (P < 0.05). However, higher apparent BMD and more compact plate-like trabeculae were observed in the mandible after treatment with zoledronate as compared to the findings in the proximal tibia. No significant differences were found in any parameter in both zoledronate groups. Conclusions  The present study showed that zoledronate could reverse the significant bone loss in mice mandibles that was induced by OPG gene deficiency. This preventive effect, which was accompanied with considerable inhibition of bone turnover, differed in the mandible and in the proximal tibia and was independent of the OPG pathway. Drs. Sheng and Xu contributed equally to this work.     

An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of dysregulated cellular differentiation characterized by malformation of the great toes during embryonic skeletal development and by progressive heterotopic endochondral ossification postnatally. Patients with these classic clinical features of FOP have the identical heterozygous single nucleotide substitution (c.617G > A; R206H) in the gene encoding ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Gene targeting was used to develop an Acvr1 knock‐in model for FOP (Acvr1^R206H/+). Radiographic analysis of Acvr1^R206H/+ chimeric mice revealed that this mutation induced malformed first digits in the hind limbs and postnatal extraskeletal bone formation, recapitulating the human disease. Histological analysis of murine lesions showed inflammatory infiltration and apoptosis of skeletal muscle followed by robust formation of heterotopic bone through an endochondral pathway, identical to that seen in patients. Progenitor cells of a Tie2⁺ lineage participated in each stage of endochondral osteogenesis. We further determined that both wild‐type (WT) and mutant cells are present within the ectopic bone tissue, an unexpected finding that indicates that although the mutation is necessary to induce the bone formation process, the mutation is not required for progenitor cell contribution to bone and cartilage. This unique knock‐in mouse model provides novel insight into the genetic regulation of heterotopic ossification and establishes the first direct in vivo evidence that the R206H mutation in ACVR1 causes FOP. © 2012 American Society for Bone and Mineral Research.     

Bisphosphonate Inhibits Bone Turnover in OPG−/− Mice Via a Depressive Effect on Both Osteoclasts and Osteoblasts

Osteoclast differentiation and functioning are strictly controlled by RANKL expressed on osteoblast membrane surfaces, but whether osteoclasts exert control over osteoblasts remains unclear. In the present study, we examined the effect of an osteoclast inhibitor, a bisphosphonate (BP), on the response of maxillary bone to mechanical stress in a high-turnover osteoporosis model (OPG^−/− mice, a model of juvenile Paget disease). Mechanical stress was induced by use of orthodontic elastics to move the maxillary first molar. BP was administered once per day beginning 5 days before elastic insertion. Relative to wild type (WT), in the OPG^−/− mice tooth movement distance was greater, resorption of the interradicular septum occurred to a greater extent, the osteoclast count was higher, and serum alkaline phosphatase (ALP) was higher. However, administration of BP to OPG^−/− mice reduced tooth movement distance, increased bone volume at the interradicular septum, decreased the osteoclast count, and reduced serum ALP. BP administration also caused a temporal shift in peak Runx2 staining in OPG^−/− mice, such that the overall staining time course was similar to that observed for WT mice. We conclude that BP administration not only inhibited osteoclast activity in OPG^−/− mice but also systemically and locally inhibited osteoblast activity. It is possible that osteoclasts are able to exert some negative control over osteoblasts.     

Genetic and pharmacological inhibition of retinoic acid receptor γ function promotes endochondral bone formation

The nuclear retinoic acid receptors (RARs) play key roles in skeletal development and endochondral ossification. Previously, we showed that RARγ regulates chondrogenesis and that pharmacological activation of RARγ blocked heterotopic ossification (HO), pathology in which endochondral bone forms in soft tissues. Thus, we reasoned that pharmacological inhibition of RARγ should enhance endochondral ossification, leading to a potential therapeutic strategy for bone deficiencies. We created surgical bone defects in wild type and RARγ‐null mice and monitored bone healing. Fibrous, cartilaginous, and osseous tissues formed in both groups by day 7, but more cartilaginous tissue formed in mutants within and around the defects compared to controls. Next, we implanted a mixture of Matrigel and rhBMP2 subdermally to induce ectopic endochondral ossification. Administration of RARγ antagonists significantly stimulated ectopic bone formation in wild type but not in RARγ‐null mice. The antagonist‐induced increases in bone formation were preceded by increases in cartilage formation and were accompanied by higher levels of phosphorylated Smad1/5/8 (pSmad1/5/8) compared to vehicle‐treated control. Higher pSmad1/5/8 levels were also observed in cartilaginous tissues forming in healing bone defects in RARγ‐null mice, and increases in pSmad1/5/8 levels and Id1‐luc activity were observed in RARγ antagonist‐treated chondrogenic cells in culture. Our data show that genetic or pharmacological interference with RARγ stimulates endochondral bone formation and does so at least in part by stimulating canonical BMP signaling. This pharmacologic strategy could represent a new tool to enhance endochondral bone formation in the setting of various orthopedic surgical interventions and other skeletal deficiencies. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1096–1105, 2017.

     

Skeletal Analysis of the Long Bone Abnormality (lbab/lbab) Mouse, A Novel Chondrodysplastic C-Type Natriuretic Peptide Mutant

Long bone abnormality (lbab/lbab) is a strain of dwarf mice. Recent studies revealed that the phenotype is caused by a spontaneous mutation in the Nppc gene, which encodes mouse C-type natriuretic peptide (CNP). In this study, we analyzed the chondrodysplastic skeletal phenotype of lbab/lbab mice. At birth, lbab/lbab mice are only slightly shorter than their wild-type littermates. Nevertheless, lbab/lbab mice do not undergo a growth spurt, and their final body and bone lengths are only ~60% of those of wild-type mice. Histological analysis revealed that the growth plate in lbab/lbab mice, especially the hypertrophic chondrocyte layer, was significantly thinner than in wild-type mice. Overexpression of CNP in the cartilage of lbab/lbab mice restored their thinned growth plate, followed by the complete rescue of their impaired endochondral bone growth. Furthermore, the bone volume in lbab/lbab mouse was severely decreased and was recovered by CNP overexpression. On the other hand, the thickness of the growth plate of lbab/+ mice was not different from that of wild-type mice; accordingly, impaired endochondral bone growth was not observed in lbab/+ mice. In organ culture experiments, tibial explants from fetal lbab/lbab mice were significantly shorter than those from lbab/+ mice and elongated by addition of 10⁻⁷ M CNP to the same extent as lbab/+ tibiae treated with the same dose of CNP. These results demonstrate that lbab/lbab is a novel mouse model of chondrodysplasia caused by insufficient CNP action on endochondral ossification.     

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes osteoclastogenesis via induction of and interaction with dendritic cell–specific transmembrane protein (DC‐STAMP)

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF‐κB ligand (GST‐RANKL), and a combination of recombinant CCN2 (rCCN2) and GST‐RANKL significantly enhanced tartrate‐resistant acid phosphatase (TRACP)–positive multinucleated cell formation compared with GST‐RANKL alone. Therefore, we suspected the involvement of CCN2 in cell‐cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real‐time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid‐phase binding assay of CCN2 and dendritic cell–specific transmembrane protein (DC‐STAMP), which is involved in cell‐cell fusion. The results showed that CCN2 induced and interacted with DC‐STAMP. Furthermore, GST‐RANKL–induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC‐STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC‐STAMP. © 2011 American Society for Bone and Mineral Research.     

Reversibility of alendronate-induced contraction in human osteoclast-like cells formed from bone marrow cells in culture

Alendronate is a powerful therapeutic agent for the treatment of hypercalcemia in malignancy and osteoporosis and has recently been developed as a treatment for hypercalcemia of malignancy. In this study, time-lapse cinemicrography was used to investigate the effects of this agent on the morphology and the motility of human osteoclast-like multinucleated cells (MNCs) from human bone marrow. Alendronate at 10⁻⁵ M induced contraction of the cells starting 7.5 h after its addition. contraction was markedly induced immediately after alendronate removal. However, contraction almost disappeared 18 h after removal, and osteoclast-like MNCs recovered their original sizes and shape. There was only partial recovery from contraction after alendronate treatment at 10⁻⁴ M. In contrast, untreated control cells did not change their morphology after washing with culture medium. Motility analysis showed that osteoclast-like MNCs treated with 10⁻⁵ M alendronate moved actively after washing, but at 10⁻⁴ M the motility locus was very narrow. At 10⁻⁴ M, the actin ring in the cells began to break down, beginning 6 h after addition. The effects of alendronate on human osteoclast-like MNCs morphology and motility were reversible at 10⁻⁵ M, suggesting that alendronate dose not cause any cellular damages in human osteoclasts up to 10⁻⁵ M, which is an effective dose for bone resorption. Received: June 18, 1998/Accepted: Oct. 30, 1998     

Gab2 plays distinct roles in bone homeostasis at different time points

Grb2-associated binder 2 (Gab2) is an adaptor molecule that can be tyrosine phosphorylated by various growth factors and cytokines. Gab2 is known to play a role in signaling pathways downstream of cytokines that regulate bone homeostasis, including M-CSF, RANKL, and IL-6. To clarify the role of Gab2 in bone homeostasis during distinct phases of skeletal development, we compared phenotypic changes in bone homeostasis in Gab2 ^−/− mice at two different ages. Although Gab2 ^−/− mice showed increased bone volume at both time points, the reasons underlying the increased bone volume differed. At 6 weeks, the increased bone volume was due to enhanced bone resorption and bone formation, indicating that Gab2 plays a negative regulatory role for both osteoclastogenesis and osteoblast differentiation. At 12 weeks, the increased bone volume resulted from reduced osteoclast differentiation, indicating that Gab2 plays a positive regulatory role for osteoclastogenesis. Thus, Gab2 plays opposite roles in osteoclastogenesis during the phases of skeletal development and maintenance.     

Smad6 is essential to limit BMP signaling during cartilage development

Bone morphogenetic protein (BMP) signaling pathways regulate multiple aspects of endochondral bone formation. The importance of extracellular antagonists as regulators of BMP signaling has been defined. In vitro studies reveal that the intracellular regulators, inhibitory Smads 6 and 7, can regulate BMP‐mediated effects on chondrocytes. Although in vivo studies in which inhibitory Smads were overexpressed in cartilage have shown that inhibitory Smads have the potential to limit BMP signaling in vivo, the physiological relevance of inhibitory Smad activity in skeletal tissues is unknown. In this study, we have determined the role of Smad6 in endochondral bone formation. Loss of Smad6 in mice leads to defects in both axial and appendicular skeletal development. Specifically, Smad6?/? mice exhibit a posterior transformation of the seventh cervical vertebra, bilateral ossification centers in lumbar vertebrae, and bifid sternebrae due to incomplete sternal band fusion. Histological analysis of appendicular bones revealed delayed onset of hypertrophic differentiation and mineralization at midgestation in Smad6?/? mice. By late gestation, however, an expanded hypertrophic zone, associated with an increased pool of proliferating cells undergoing hypertrophy, was evident in Smad6 mutant growth plates. The mutant phenotype is attributed, at least in part, to increased BMP responsiveness in Smad6‐deficient chondrocytes. Overall, our results show that Smad6 is required to limit BMP signaling during endochondral bone formation. © 2011 American Society for Bone and Mineral Research