Porphyromonas gingivalis infection increases osteoclastic bone resorption and osteoblastic bone formation in a periodontitis mouse model

Authors

Wenjian Zhang, Jun Ju, Todd Rigney and Gena Tribble

Abstract

Background Porphyromonas gingivalis has been shown to invade osteoblasts and inhibit their differentiation and mineralization in vitro. However, it is unclear if P. gingivalis can invade osteoblasts in vivo and how this would affect alveolar osteoblast/osteoclast dynamics. This study aims to answer these questions using a periodontitis mouse model under repetitive P. gingivalis inoculations.

Methods For 3-month-old BALB/cByJ female mice, 109 CFU of P. gingivalis were inoculated onto the gingival margin of maxillary molars 4 times at 2-day intervals. After 2 weeks, another 4 inoculations at 2-day intervals were applied. Calcein was injected 7 and 2 days before sacrificing animals to label the newly formed bone. Four weeks after final inoculation, mice were sacrificed and maxilla collected. Immunohistochemistry, micro-CT, and bone histomorphometry were performed on the specimens. Sham infection with only vehicle was the control.

Results P. gingivalis was found to invade gingival epithelia, periodontal ligament fibroblasts, and alveolar osteoblasts. Micro-CT showed alveolar bone resorption and significant reduction of bone mineral density and content in the infected mice compared to the controls. Bone histomorphometry showed a decrease in osteoblasts, an increase in osteoclasts and bone resorption, and a surprisingly increased osteoblastic bone formation in the infected mice compared to the controls.

Conclusions P. gingivalis invades alveolar osteoblasts in the periodontitis mouse model and cause alveolar bone loss. Although P. gingivalis appears to suppress osteoblast pool and enhance osteoclastic bone resorption, the bone formation capacity is temporarily elevated in the infected mice, possibly via some anti-microbial compensational mechanisms.

Link To Article

http://dx.doi.org/10.1186/1472-6831-14-89

A Combination of rhBMP-2 (Recombinant Human Bone Morphogenetic Protein-2) and MEK (MAP Kinase/ERK Kinase) Inhibitor...

Title

A Combination of rhBMP-2 (Recombinant Human Bone Morphogenetic Protein-2) and MEK (MAP Kinase/ERK Kinase) Inhibitor PD0325901 Increases Bone Formation in a Murine Model of Neurofibromatosis Type I Pseudarthrosis

Authors

J. El-Hoss, PhD; T. Cheng, BE/BMedSc; E.C. Carpenter, MRCS; K. Sullivan, PhD; N. Deo, BSc(Hons); K. Mikulec ; D.G. Little, MBBS, FRACS(Orth), PhD; A. Schindeler, PhD

Abstract

Background: Congenital tibial dysplasia is a severe pediatric condition that classically results in a persistent pseudarthrosis. A majority of these cases are associated with neurofibromatosis type I (NF1), a genetic disorder in which inactivation of the NF1 gene leads to overactivity of the Ras-MEK-MAPK (mitogen-activated protein kinase) signaling pathway. We therefore hypothesized that pharmaceutical inhibition of MEK-MAPK may be a beneficial therapeutic strategy.

Methods: In vitro methods were used to demonstrate a role for the MEK inhibitor PD0325901 in promoting osteogenic differentiation in Nf1−/− calvarial osteoblasts. Local applications of rhBMP-2 and/or PD0325901 were then tested in a mouse model of NF1 tibial pseudarthrosis featuring localized double inactivation of the Nf1 gene in a fracture. Mice received no treatment, PD0325901 (10 mg/kg/day from two days before fracture to ten days after fracture), rhBMP-2 (10 μg), or a combination of rhBMP-2 and PD0325901.

Results: Animals treated with the delivery vehicle alone, PD0325901, rhBMP-2, or the PD0325901 + rhBMP-2 combination showed union rates of 0%, 8%, 69% (p < 0.01), or 80% (p < 0.01), respectively, at twenty-one days after fracture. Mice treated with the rhBMP-2 + PD0325901 combination displayed a callus volume sixfold greater than the vehicle controls and twofold greater than the group receiving rhBMP-2 alone. Although MEK inhibition combined with rhBMP-2 led to increases in bone formation and union, the proportion of fibrous tissue in the callus was not significantly reduced.

Conclusions: The data suggest that MEK inhibition can promote bone formation in combination with rhBMP-2 in the context of an NF1 pseudarthrosis. However, PD0325901 did not promote substantive bone anabolism in the absence of an exogenous anabolic stimulus and did not suppress fibrosis.

Clinical Relevance: This study examines a signaling pathway-based approach to treating poor bone healing in a model of NF1 pseudarthrosis.

Link To Article

http://dx.doi.org/10.2106/JBJS.M.00862

Estrogen via Estrogen Receptor Beta Partially Inhibits Mandibular Condylar Cartilage Growth

Authors

J. Chen, Y. Kamiya, I. Polur, M. Xu, T. Choi, Z. Kalajzic, H. Drissi, S. Wadhwa

Abstract

Objective Temporomandibular joint diseases predominantly afflict women, suggesting a role for female hormones in the disease process. However, little is known about the role of estrogen receptor (ER) signaling in regulating mandibular condylar cartilage growth. Therefore, the goal of this study was to examine the effects of altered estrogen levels on the mandibular condylar cartilage in WT and ER beta KO mice.

Materials and Methods 21-day-old female WT (n=37) and ER beta KO mice (n=36) were either sham operated or ovariectomized, and treated with either placebo or estradiol. The mandibular condylar cartilage was evaluated by histomorphometry, proliferation was analyzed by double EdU/BrdU labeling, and assays on gene and protein expression of chondrocyte maturation markers were performed.

Results In WT mice, ovariectomy caused a significant increase in mandibular condylar cartilage cell numbers, a significant increase in Sox9 expression and a significant increase in proliferation compared with sham operated WT mice. In contrast, ovariectomy did not cause any of these effects in the ER beta KO mice. Estrogen replacement treatment in ovariectomized WT mice caused a significant decrease in ER alpha expression and a significant increase in Sost expression compared with ovariectomized mice treated with placebo. Estrogen replacement treatment in ovariectomized ER beta KO mice caused a significant increase in Col2 expression, no change in ER alpha expression, and a significant increase in Sost expression.

Conclusion Estrogen via ER beta inhibits proliferation and ER alpha expression while estrogen independent of ER beta induces Col2 and Sost expression.

Link To Article

http://dx.doi.org/10.1016/j.joca.2014.07.003

Loss of Cbl–PI3K interaction in mice prevents significant bone loss following ovariectomy

Authors

Naga Suresh Adapala, Danielle Holland, Vanessa Scanlon, Mary F. Barbe, Wallace Y. Langdon, Alexander Y. Tsygankov, Joseph A. Lorenzo, Archana Sanjay

Abstract

Cbl and Cbl-b are E3 ubiquitin ligases and adaptor proteins, which perform regulatory roles in bone remodeling. Cbl−/− mice have delayed bone development due to decreased osteoclast migration. Cbl-b−/− mice are osteopenic due to increased bone resorbing activity of osteoclasts. Unique to Cbl, but not present in Cbl-b, is tyrosine 737 in the YEAM motif, which upon phosphorylation provides a binding site for the regulatory p85 subunit of PI3K. Substitution of tyrosine 737 with phenylalanine (Y737F, CblYF/YF mice) prevents Y737 phosphorylation and abrogates the Cbl–PI3K interaction. We have previously reported that CblYF/YF mice had increased bone volume due to defective bone resorption and increased bone formation. Here we show that the lumbar vertebra from CblYF/YF mice did not have significant bone loss following ovariectomy. Our data also suggests that abrogation of Cbl–PI3K interaction in mice results in the loss of coupling between bone resorption and formation, since ovariectomized CblYF/YF mice did not show significant changes in serum levels of c-terminal telopeptide (CTX), whereas the serum levels of pro-collagen type-1 amino-terminal pro-peptide (P1NP) were decreased. In contrast, following ovariectomy, Cbl−/− and Cbl-b−/− mice showed significant bone loss in the tibiae and L2 vertebrae, concomitant with increased serum CTX and P1NP levels. These data indicate that while lack of Cbl or Cbl-b distinctly affects bone remodeling, only the loss of Cbl–PI3K interaction protects mice from significant bone loss following ovariectomy.

Link To Article

http://dx.doi.org/10.1016/j.bone.2014.06.013

The mineral dissolution function of osteoclasts is dispensable for hypertrophic cartilage degradation during long bone development and growth

Authors

Heiani Touaitahuata, Gaelle Cres, Sylvain de Rossi, Virginie Vives, Anne Blangy

Abstract

During long bone development and post-natal growth, the cartilaginous model of the skeleton is progressively replaced by bone, a process known as endochondral ossification. In the primary spongiosa, osteoclasts degrade the mineralized cartilage produced by hypertrophic chondrocytes to generate cartilage trabeculae that osteoblasts embed in bone matrix. This leads to the formation of the trabecular bone network of the secondary spongiosa that will undergo continuous remodeling. Osteoclasts are specialized in mineralized tissue degradation, with the combined ability to solubilize hydroxyapatite and to degrade extracellular matrix proteins. We reported previously that osteoclasts lacking Dock5 could not degrade bone due to abnormal podosome organization and absence of sealing zone formation. Consequently, adult Dock5−/− mice have increased trabecular bone mass. We used Dock5−/− mice to further investigate the different functions of osteoclast during endochondral bone formation. We show that long bones are overall morphologically normal in developing and growing Dock5−/− mice. We demonstrate that Dock5−/− mice also have normal hypertrophic cartilage and cartilage trabecular network. Conversely, trabecular bone volume increased progressively in the secondary spongiosa of Dock5−/− growing mice as compared to Dock5+/+ animals, even though their osteoclast numbers were the same. In vitro, we show that Dock5−/− osteoclasts do present acidic compartments at the ventral plasma membrane and produce normal amounts of active MMP9, TRAP and CtsK for matrix protein degradation but they are unable to solubilize minerals. These observations reveal that contrarily to bone resorption, the ability of osteoclasts to dissolve minerals is dispensable for the degradation of mineralized hypertrophic cartilage during endochondral bone formation.

Link To Article

http://dx.doi.org/10.1016/j.ydbio.2014.06.020

Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1

Authors

Jean de la Croix Ndong, Alexander J Makowski, Sasidhar Uppuganti, Guillaume Vignaux, Koichiro Ono, Daniel S Perrien, Simon Joubert, Serena R Baglio, Donatella Granchi, David A Stevenson, Jonathan J Rios, Jeffry S Nyman & Florent Elefteriou

Abstract

Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase–activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal–regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2–induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Link To Article

http://dx.doi.org/10.1038/nm.3583