Meriem Lamghari

Greater Bone Formation of Y2 Knockout Mice Is Associated with Increased Osteoprogenitor Numbers and Altered Y1 Receptor Expression

Germ line or hypothalamus-specific deletion of Y2 receptors in mice results in a doubling of trabecular bone volume. However, the specific mechanism by which deletion of Y2 receptors increases bone mass has not yet been identified. Here we show that cultured adherent bone marrow stromal cells from Y2-/- mice also demonstrate increased mineralization in vitro. Isolation of two populations of progenitor cell types, an immature mesenchymal stem cell population and a more highly differentiated population of progenitor cells, revealed a greater number of the progenitor cells within the bone of Y2-/- mice. Analysis of Y receptor transcripts in cultured stromal cells from wild-type mice revealed high levels of Y1 but not Y2, Y4, Y5, or y6 receptor mRNA. Interestingly, germ line Y2 receptor deletion causes Y1 receptor down-regulation in stromal cells and bone tissue possibly due to the lack of feedback inhibition of NPY release and subsequent overstimulation of Y1 receptors. Furthermore, deletion of Y1 receptors resulted in increased bone mineral density in mice. Together, these findings indicate that the greater number of mesenchymal progenitors and the altered Y1 receptor expression within bone cells in the absence of Y2 receptors are a likely mechanism for the greater bone mineralization in vivo and in vitro, opening up potential new treatment avenues for osteoporosis.

Stimulation of bone marrow cells and bone formation by nacre: in vivo and in vitro studies

There is frequently a loss of vertebral bone due to disease or aging. Nacre (mother of pearl from the oyster Pinctada maxima) stimulates bone cell differentiation and bone formation in vitro and in vivo. Experimental bone defects were prepared in the vertebrae of sheep and used to test the suitability of nacre as an injectable osteogenic biomaterial for treating vertebral bone loss. Twenty-one cavities were prepared in the first four upper lumbar vertebrae of 11 sheep and filled with nacre powder. The lumbar vertebrae were removed after 1 to 12 weeks, embedded undecalcified in methacrylate, and processed for histological studies. The nacre slowly dissolved and the experimental cavities contained a large active cell population. By 12 weeks, the experimental cavity was occupied by newly matured bone trabeculae in contact with or adjacent to the dissolving nacre. The functional new bone trabeculae were covered with osteoid lined with osteoblasts, indicating continuing bone formation. The in vitro study on rat bone marrow explants cultured with a water-soluble extract of the nacre organic matrix also resulted in the stimulation of osteogenic bone marrow cells with enhanced alkaline phosphatase activity. Thus, both the in vivo and in vitro findings suggest that nacre contains one or more signal molecules capable of activating osteogenic bone marrow cells.

Bone reactions to nacre injected percutaneously into the vertebrae of sheep.

We have studied the osteogenic effects of nacre (mother of pearl) placed in experimental cavities prepared in the lumbar vertebrae of sheep. Some of cavities were filled with nacre, some with PMMA, and some were left empty. The vertebrae were removed 1, 8, 12 weeks after surgery, and assessed histologically and morphometrically. The nacre particles in the bone cavity and the surrounding intertrabecular spaces gradually dissolved beginning at 8 weeks after surgery. There were layers of newly formed bone, both woven and lamellar, in various stages of maturation in contact with or adjacent to the dissolving nacre. Quantitative assessment of the activation of bone formation adjacent to the cavities filled with nacre indicated significant activation of bone formation, which continued until week 12. There was also increased mineralization of the host bone at this time. There was no new bone formation in the empty cavities, or in those filled with PMMA. PMMA also caused necrosis of surrounding bone cells with a change in bone architecture and significant reductions in bone formation and mineralization. This study demonstrates that nacre stimulates bone-forming cells in vertebrae and appears to result in new bone formation.

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts.

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3‐E1 osteoblasts using RT‐PCR and ELISA measurements. We have at first identified the expression of Ob‐Rb and Ob‐Ra leptin receptor isoforms in MC3T3‐E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose‐dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3‐E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene. J. Cell. Biochem. 98: 1123–1129, 2006.

NPY revealed as a critical modulator of osteoblast function in vitro: New insights into the role of Y1 and Y2 receptors

Neuropeptide Y (NPY) has recently emerged as a potential regulator of bone homeostasis. However, the relevance of NPYs role in osteoblast activity and the biological functions involving NPY receptors in bone homeostasis remain to be clarified. Here we report that chronically elevated NPY levels leaded to a modulation of the level of Y2 receptor expression marked with a transient down and upregulation according to the stage of osteoblast differentiation. We also show that NPY is a negative regulator of Y1 receptor expression. The pharmalogical activation of Y2 receptor with its agonist resulted in similar effect. Functional analysis also revealed the osteogenic potential of NPY with osteoblast phenotype markers being significantly enhanced in osteoprogenitor cells stimulated by NPY, probably due to the down‐regulation of Y1 receptor. In contrasts, these cells exhibit a reduction in calcium deposition in extracellular matrix most likely mediated via Y2 receptor signalling. Furthermore, we show that NPY modulates receptor activator of nuclear factor kB (NF‐kB) (RANK) ligand and osteoprotegerin, two key factors regulating bone remodelling. Specifically, NPY inhibits the transcriptional activity of RANKL promoter in osteoprogenitor cells and enhances OPG expression in osteoblasts at early stages of differentiation. However, NPY effect on OPG seemed to be unrelated to Y2 receptor activation. Taken together the present data supported the contribution of NPY pathway in bone homeostasis via a direct action on osteoblasts cells. J. Cell. Biochem. 107: 908–916, 2009.

Neuropeptide Y and osteoblast differentiation - the balance between the neuro-osteogenic network and local control

Accumulating evidence has contributed to a novel view in bone biology: bone remodeling, specifically osteoblast differentiation, is under the tight control of the central and peripheral nervous systems. Among other players in this neuro‐osteogenic network, the neuropeptide Y (NPY) system has attracted particular attention. At the central nervous system level, NPY exerts its function in bone homeostasis through the hypothalamic Y2 receptor. Locally in the bone, NPY action is mediated by its Y1 receptor. Besides the presence of Y1, a complex network exists locally: not only there is input of the peripheral nervous system, as the bone is directly innervated by NPY‐containing fibers, but there is also input from non‐neuronal cells, including bone cells capable of NPY expression. The interaction of these distinct players to achieve a multilevel control system of bone homeostasis is still under debate. In this review, we will integrate the current knowledge on the impact of the NPY system in bone biology, and discuss the mechanisms through which the balance between central and the peripheral NPY action might be achieved.

Neuropeptide Y Y1 receptor antagonism increases bone mass in mice.

The neuropeptide Y system has emerged as one of the major neural signalling pathways regulating bone homeostasis. Absence of Y1 receptor signalling from bone forming osteoblasts is responsible for an enhancement on bone mass in mice, suggesting that pharmacological blockade of Y1 receptors may offer a novel anabolic treatment option for improving bone mass. Here we show that oral administration of the selective Y1 receptor antagonist BIBO3304 for 8 weeks dose-dependently increases bone mass in mice. Histomorphometric analysis revealed a significant 1.5-fold increase in cancellous bone volume in the femora of mice treated with BIBO3304. Furthermore, bone microarchitecture was improved, with greater trabecular number and trabecular thickness. This increase in bone mass was associated with a significant increase in bone anabolic activity of osteoblasts and, interestingly, was evident despite a coincident increase in bone resorption, as evidenced by an increase in the number of the osteolytic osteoclasts. Changes were also evident in cortical bone, with a significant increase in periosteal mineral apposition rate. Importantly, no adverse extra-skeletal side effects were observed through Y1 receptor antagonism over the 8-week treatment period, with no effects of even the higher BIBO3304 dose on body weight, adiposity, energy metabolism or circulating corticosterone levels. Taken together, this work describes the first NPY-based anabolic treatment for improving bone mass, and highlights the therapeutic potential of blocking Y1 receptor signalling for the prevention of, or recovery from, degenerative skeletal diseases.

Arthrodesis of lumbar spine transverse processes using nacre in rabbit.

This study compares the osteogenic effects of nacre and autogenous bone grafts in a rabbit model of lumbar spine transverse process arthrodesis. A total of 15 rabbits were processed for arthrodesis between the fifth and sixth lumbar vertebrae using nacre powder mixed with autologous blood or autogenous iliac crest bone. Control rabbits were sham operated. Sample vertebrae were removed from the nacre‐implanted rabbits at 2, 5, and 11 weeks postsurgery. The autogenous bone graft and sham‐operated groups were processed for histological study 11 weeks postsurgery. The results for the three groups were compared at 11 weeks. The nacre‐implanted samples taken at 2 weeks showed that the nacre was well tolerated by the host tissue. Endochondral bone formation was seen in the region of the dissolving nacre particles by 5 weeks. The newly formed bone formed a solid fusion between the transverse processes in one‐third of the rabbits. There was still new bone formation at 11 weeks at the nacre implant site. Two‐thirds of the rabbits had formed a solid fusion. Light microscopy also showed new bone formation 11 weeks after the autologous bone graft. All rabbits had a solid fusion. This initial study indicates that nacre can induce spinal fusion in an acceptable percentage of cases.

Adsorbed fibrinogen leads to improved bone regeneration and correlates with differences in the systemic immune response

Designing new biomaterials that can modulate the inflammatory response instead of attempting just to reduce it constitutes a paradigm change in regenerative medicine. This work aimed to investigate the capacity of an immunomodulatory biomaterial to enhance bone regeneration. For that purpose we incorporated a molecule with well-established pro-inflammatory and pro-healing roles, fibrinogen, in chitosan scaffolds. Two different incorporation strategies were tested, leading to concentrations of 0.54±0.10mg fibrinogen g(-1) scaffold immediately upon adsorption (Fg-Sol), and 0.34±0.04mg fibrinogen g(-1) scaffold after washing (Fg-Ads). These materials were implanted in a critical size bone defect in rats. At two months post-implantation the extent of bone regeneration was examined by histology and the systemic immune response triggered was evaluated by determining the percentages of myeloid cells, T and B lymphocytes in the draining lymph nodes. The results obtained indicate that the fibrinogen incorporation strategy conditioned the osteogenic capacity of biomaterials. Fg-Ads scaffolds led to more bone formation, and the presence of Fg stimulated angiogenesis. Furthermore, animals implanted with Fg-Ads scaffolds showed significant increases in the percentages of B lymphocytes and myeloid cells in the draining lymph nodes, while levels of T lymphocytes were not significantly different. Finally, a significant increase in TGF-β1 was detected in the plasma of animals implanted with Fg-Ads. Taken together the results presented suggest a potential correlation between the elicited immune response and biomaterial osteogenic performance.

A model for evaluating injectable bone replacements in the vertebrae of sheep: radiological and histological study

We developed a bone-defect model in the vertebrae of sheep. Forty four cavities were prepared in the upper lumbar vertebrae of 11 sheep using a biopsy trocar via a posterior-lateral extracanal percutaneous route and the location was monitored by radiology with a brilliance amplifier. The cavities were 3 mm in diameter. The histological study was performed on 15 cavities which were left empty to give reference data for the model. Histological and histomorphometry results showed that 67% of the surface area of the empty cavities was still empty 3 months after their preparation. Thus, the natural regenerative capacity of vertebral trabecular bone is limited. We performed preliminary percutaneous injections of polymethylmethacrylate (PMMA) and nacre powder to assess whether this bone-defect model would be suitable for further studies on bone repair. Cavities were successfully filled with nacre powder (21 cavities) or PMMA (8 cavities) while monitoring the process by interventional radiology. The experimental sheep vertebrae defect system is reproducible and appears to be a suitable model for testing injectable biomaterials for treating bone loss.